Fagopyritol synthase genes and uses thereof

ABSTRACT

The present invention relates to an isolated DNA molecule encoding a fagopyritol synthase. A method for producing a fagopyritol, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor is also described. The method includes providing a fagopyritol synthase, providing a substrate comprising a galactosyl donor and a galactosyl acceptor, and combining the fagopyritol synthase with the substrate under conditions effective produce a fagopyritol, an insulin mediator, an insulin mediator analogue, an insulin mediator homologue, or an insulin mediator inhibitor.

This application is a continuation of U.S. patent application Ser. No.14/104,788, filed Dec. 12, 2013, which is a divisional of U.S. patentapplication Ser. No. 12/785,097, filed May 21, 2010, which is adivisional of U.S. patent application Ser. No. 10/435,226, filed May 9,2003, now U.S. Pat. No. 7,807,406, issued Oct. 5, 2010, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 60/379,373,filed May 9, 2002, which are hereby incorporated by reference in theirentirety.

The present invention was developed with support under Cooperative StateResearch, Education and Extension Service, U.S. Department ofAgriculture Project No. NYC-125323. The U.S. Government has certainrights.

FIELD OF THE INVENTION

The present invention relates to fagopyritol synthase genes and methodsof producing fagopyritols, insulin mediators, insulin mediatoranalogues, or insulin mediator homologues.

BACKGROUND OF THE INVENTION

Embryos of many plant seeds accumulate sucrose and the raffinose familyof oligosaccharides (RSO), such as raffinose, stachyose and verbascose,as the major soluble sugars in mature seeds (Horbowicz et al., Seed Sci.Res. 4:385-405 (1994); Obendorf, See Sci. Res. 7:63-74 (1997)). Soybean(Glycine max (L.) Merrill) seeds accumulate soluble carbohydrates,primarily sucrose, raffinose, and stachyose and lesser amounts ofgalactopinitol A, galactopinitol B, ciceritol, and fagopyritol B1 inaxis and cotyledon tissues as part of the seed maturation process(Obendorf et al., Crop Science 38:78-84 (1998)). By contrast, embryos ofmaturing buckwheat (Fagopyrum esculentum Moench) seeds accumulatefagopyritols, galactosyl derivatives of D-chiro-inositol, instead ofraffinose and stachyose (Horbowicz et al., Planta 205:1-11 (1998)). Sixfagopyritols, in two different series, are present in buckwheat embryos:fagopyritol A1 (α-D-galactopyranosyl-(1→3)-1D-chiro-inositol),fagopyritol A2(α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol),fagopyritol A3(α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol),fagopyritol B1 (α-D-galactopyranosyl-(1→2)-1D-chiro-inositol),fagopyritol B2(α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→2)-1D-chiro-inositol),and fagopyritol B3(α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→2)-1D-chiro-inositol)(Horbowicz et al., Planta 205:1-11 (1998); Szczecinski et al., Bull.Polish Acad. Sci., Chem. 46:9-13 (1998); Obendorf et al., Carbohydr.Res. 328:623-627 (2000); Steadman et al., Carbohydr. Res. 331:19-25(2001)). Fagopyritols are concentrated in the axis and cotyledon tissuesof embryos in mature buckwheat seeds (Horbowicz et al., Planta 205:1-11(1998)). Buckwheat bran, a commercial milling fraction (Steadman et al.,J. Cereal Sci. 33:271-278 (2001)), is a rich source of fagopyritols(Steadman et al., J. Agric. Food Chem. 48:2843-2847 (2000)).

Fagopyritols are of considerable interest for the treatment ofnon-insulin dependent diabetes mellitus (NIDDM) and polycystic ovarysyndrome (PCOS), both insulin response disorders. Fagopyritol A1 isisosteric with2-amino-2-deoxy-α-D-galactopyranosyl-(1→3)-1D-chiro-inositol (Berlin etal., Tetrahedron Lett. 31:1109-1112 (1990)) related to a putativeinsulin mediator (Berlin et al., Tetrahedron Lett. 31:1109-1112 (1990);Lamer et al., Biochem. Biophys. Res. Comm. 151:1416-1426 (1988))deficient in subjects with NIDDM (Fonteles et al., Diabetologia39:731-734 (1996); Lamer et al., Diabetes Rev. 7:217-231 (1999)) andPCOS (Nestler et al., J. Clin. Endocrin. Metab. 83:2001-2005 (1998);Nestler et al., New England J. Med. 340:1314-1320 (1999); Nestler etal., J. Pediatric Endocrin. Metab. 13(Suppl. 5):1295-1298 (2000)).

Enzymes (fagopyritol synthases) catalyzing the biosynthesis offagopyritols in buckwheat or other plants have not been described. Thepresent invention is directed to overcoming this and other deficienciesin the prior art.

SUMMARY OF THE INVENTION

The present invention relates to isolated nucleic acid molecules whichencode a fagopyritol synthase and the amino acid sequences encoded bysuch nucleic acid molecules.

Another aspect of the present invention pertains to host cells,expression vectors, transgenic plants, and transgenic plant seedscontaining the isolated nucleic acid molecules of the present invention.

The present invention is also directed to a method for producing afagopyritol, an insulin mediator, an insulin mediator analogue, or aninsulin mediator homologue. This method includes providing a fagopyritolsynthase, providing a substrate including a galactosyl donor and agalactosyl acceptor, and combining the fagopyritol synthase with thesubstrate under conditions effective to produce a fagopyritol, aninsulin mediator, an insulin mediator analogue, or an insulin mediatorhomologue.

The fagopyritol synthases of the present invention can be used toproduce fagopyritols, insulin mediators, insulin mediator analogues, orinsulin mediator homologues which can be used in a pharmaceuticalcomposition which also includes a pharmaceutical carrier. Thispharmaceutical composition or, alternatively, the fagopyritols, insulinmediators, insulin mediator analogues, or insulin mediator homologuescan be administered to a patient to treat disorders, such as diabetesand PCOS. In addition, the fagopyritol synthases can be used to producetransgenic plants useful for nutraceutical applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the complete nucleotide sequence of the full-lengthFeGolS-1 cDNA clone (SEQ ID NO:1). The amino acid sequence deduced fromthe major open reading frame of the cDNA sequence is shown below (SEQ IDNO:2). The translation start (ATG) and termination (TAA) codons areunderlined.

FIG. 2 shows the complete nucleotide sequence of the full-lengthFeGolS-2 cDNA clone (SEQ ID NO:3). The amino acid sequence deduced fromthe major open reading frame of the cDNA sequence is shown below (SEQ IDNO:4). The translation start (ATG) and termination (TGA) codons areunderlined.

FIG. 3 shows the complete nucleotide sequence of the partial FeGolS-3cDNA clone (SEQ ID NO:5). The amino acid sequence deduced from the majoropen reading frame of the cDNA sequence is shown below (SEQ ID NO:6).The termination (TGA) codon is underlined.

FIG. 4 shows the complete nucleotide sequence of the soybean GmGolSclone (SEQ ID NO:7). The amino acid sequence deduced from the major openreading frame of the cDNA sequence is shown below (SEQ ID NO:8). Thetranslation start (ATG) and termination (TAA) codons are underlined.

FIG. 5 shows a summary of the cloning of three FeGolS cDNAs. The fulllength FeGolS-1 (1269 bp) and FeGolS-2 (1326 bp) cDNA clones and thepartial FeGolS-3 (986 bp) cDNA clone are diagrammed in scale with thelocations of the restriction enzyme recognition sites at the top. ForFeGolS-1 and FeGolS-2, the overlapping partial cDNA clones generated by5′ and 3′ rapid amplification of cDNA ends-polymerase chain reaction(RACE-PCR) are shown under the full-length clones. The translation start(ATG) and termination (TAA/TGA) codons are shown with their relativepositions indicated in the parentheses. The PCR primers used in theRACE-PCR assays are shown with arrows indicating the direction of thePCR amplifications.

FIG. 6 shows a multiple sequence alignment of the three FeGolS cDNAclones (protein ID: AAM96868, AAM96870, AAM96869; SEQ ID NOS:2, 4, and6, respectively). Amino acid sequences deduced from the three FeGolScDNAs were aligned by the CLUSTAL W (1.81) multiple sequence alignmentprogram. The conserved amino acid residues are shown in bold letters.The amino acid sequences corresponding to the PCR primers used in theRT-PCR assays are boxed.

FIG. 7 shows a multiple sequence alignment of GolS amino acid sequencesfrom various plant species. The amino acid sequences deduced from threeFeGolS cDNA clones (FeGolS-1 AY126718, FeGolS-2 AY126716, FeGolS-3AY126717; protein ID AAM96868, AAM96870, AAM96869; SEQ ID NOS:2, 4, and6, respectively) and G. max (SEQ. ID NO. 9) were aligned with thosereported from various plant species, including A. thaliana (SEQ ID NOS:9and 10), B. napus (SEQ ID NO:11), P. sativum (SEQ ID NO:12), O. sativa(SEQ ID NO:13), A. reptans GolS-1 (SEQ ID NO:14), and A. reptans GolS-2(SEQ ID NO:15) (indicated in the left margin), by the CLUSTAL W program.The highly conserved amino acid residues are shown in bold letters. Thehypothetical manganese-binding motif, DXD, is italicized, and anasterisk marks the conserved serine phosphorylation site. The accessionnumbers of the sequences used in the comparison are: Glycine max,AY126715 (protein ID AAM96867) (BE330777); Arabidopsis thaliana,AC002337 and AC009323; Brassica napus, AF106954; Pisum sativum,PSA243815; Ajuga reptans GolS-1, ARE237693; and Ajuga reptans GolS-2,ARE237694.

FIG. 8 shows the bacterial expression and purification of recombinantGolS proteins. The recombinant GolS proteins expressed in E. coli andsubsequently purified proteins were examined by SDS-PAGE: lane 1,protein molecular weight marker (kDa of bands indicated in the leftmargin); lanes 2 and 3, 10 μg each of the total soluble protein extractsfrom uninduced and induced bacteria cells harboring FeGolS-1 cDNA,respectively; lane 4, 0.25 μg of the purified recombinant FeGolS-1protein; lanes 5 and 6, 10 μg each of the total soluble protein extractsfrom uninduced and induced bacteria cells harboring FeGolS-2 cDNA,respectively; lane 7, 0.25 μg of the purified recombinant FeGolS-2protein; lanes 8 and 9, 10 μg each of the total soluble protein extractsfrom uninduced and induced bacteria cells harboring GmGolS cDNA,respectively; lane 10, 0.25 μg of the purified recombinant GmGolSprotein.

FIGS. 9A-F show product accumulation with purified recombinant protein.FIGS. 9A-C show fagopyritol synthase products with 20 mMD-chiro-inositol, 20 mM UDP-galactose (“UDP-Gal”), 5 mM MnCl₂, 2 mMdithiothreitol (“DTT”), and 50 mM Hepes buffer, pH 7.0. FIGS. 9D-F showgalactinol synthase products with 20 mM myo-insositol, 20 mM UDP-Gal, 5mM MnCl₂, 2 mM DTT, and 50 mM Hepes buffer, pH 7.0. Reactions were run30 to 300 minutes at 30° C. with recombinant protein FeGolS-1 (FIGS. 9Aand D), FeGolS-2 (FIGS. 9B and E), and GmGolS (FIGS. 9C and F). Productswere analyzed by high resolution gas chromatography. Retention timeswere: fagopyritol A1 (A1), 24.3 minutes; fagopyritol B1 (B1), 24.8minutes; and galactinol (Gol), 25.3 minutes.

FIGS. 10A-F are graphs showing accumulated soluble carbohydrates in axisand cotyledon tissues after precocious maturation of immature soybeanembryos as a function of myo-inositol concentration. The results areshown after feeding myo-inositol (0 to 100 mM) plus sucrose (100 to 0mM) (100 mM total concentration) for 24 hours at 25° C. followed by 14days precocious maturation in slow drying series relative humidities.Values are mean±SE (n=12). FIGS. 10A-C are axis tissues. FIGS. 10D-F arecotyledon tissues. Abbreviations in FIGS. 10A-F are as follows:myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chino),fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA),galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose(Suc).

FIG. 11 is a schematic of the proposed pathways for biosynthesis offagopyritol B1, galactinol, raffinose, stachyose, and galactopinitols.Abbreviations: Glycine max galactinol synthase (GmGolS); raffinosesynthase (RFS); stachyose synthase (STS).

FIGS. 12A-F are graphs showing accumulated soluble carbohydrates in axisand cotyledon tissues after precocious maturation of immature soybeanembryos as a function of time of slow drying. The results are shownafter feeding 30 mM myo-inositol and 100 mM sucrose for 24 hours at 25°C. followed by 0 to 14 days precocious maturation in slow drying timeseries relative humidities. Values are mean±SE (n=9). FIGS. 12A-C areaxis tissues. FIGS. 12D-F are cotyledon tissues. Abbreviations in FIGS.12A-F are as follows: myo-inositol (myo), D-pinitol (Pin),D-chiro-inositol (chiro), fagopyritol B1 (B1), galactinol (Gol),galactopinitol A (GPA), galactopinitol B (GPB), raffinose (Raf),stachyose (Sta), and sucrose (Suc).

FIGS. 13A-F are graphs showing accumulated soluble carbohydrates in axisand cotyledon tissues after precocious maturation of immature soybeanembryos as a function of D-chiro-inositol concentration. The results areshown after feeding D-chiro-inositol (0 to 100 mM) plus sucrose (100 to0 mM) (100 mM total concentration) for 24 hours at 25° C. followed by 14days precocious maturation in slow drying series relative humidities.Values are mean±SE (n=18). FIGS. 13A-C are axis tissues. FIGS. 13D-F arecotyledon tissues. Abbreviations in FIGS. 13A-F are as follows:myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro),fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA),galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose(Suc).

FIGS. 14A-F are graphs showing accumulated soluble carbohydrates in axisand cotyledon tissues after precocious maturation of immature soybeanembryos as a function of time of slow drying. The results are shownafter feeding 100 mM D-chiro-inositol for 24 hours at 25° C. followed by0 to 14 days precocious maturation in slow drying time series relativehumidities. Values are mean±SE (n=9). FIGS. 14A-C are axis tissues.FIGS. 14D-F are cotyledon tissues. Abbreviations in FIGS. 14A-F are asfollows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro),fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA),galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose(Suc).

FIGS. 15A-F are graphs showing accumulated soluble carbohydrates in axisand cotyledon tissues after precocious maturation of immature soybeanembryos as a function of D-pinitol concentration. The results are shownafter feeding D-pinitol (0 to 100 mM) plus sucrose (100 to 0 mM) (100 mMtotal concentration) for 24 hours at 25° C. followed by 14 daysprecocious maturation in slow drying series relative humidities. Valuesare mean±SE (n=9). FIGS. 15A-C are axis tissues. FIGS. 15D-F arecotyledon tissues. Abbreviations in FIGS. 15A-F are as follows:myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro),fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA),galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose(Suc).

FIGS. 16A-F are graphs showing accumulated soluble carbohydrates in axisand cotyledon tissues after precocious maturation of immature soybeanembryos as a function of time of slow drying. The results are shownafter feeding 100 mM D-pinitol for 24 hours at 25° C. followed by 0 to14 days precocious maturation in slow drying time series relativehumidities. Values are mean±SE (n=9). FIGS. 16A-C are axis tissues.FIGS. 16D-F are cotyledon tissues. Abbreviations in FIGS. 16A-F are asfollows: myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro),fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA),galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose(Suc).

FIGS. 17A-F are graphs showing accumulated soluble carbohydrates in axisand cotyledon tissues after precocious maturation of immature soybeanembryos as a function of sucrose concentration. The results are shownafter feeding sucrose (0 to 200 mM) for 24 hours at 25° C. followed by14 days precocious maturation in slow drying series relative humidities.Values are mean±SE (n=9). FIGS. 17A-C are axis tissues. FIGS. 17D-F arecotyledon tissues. Abbreviations in FIGS. 17A-F are as follows:myo-inositol (myo), D-pinitol (Pin), D-chiro-inositol (chiro),fagopyritol B1 (B1), galactinol (Gol), galactopinitol A (GPA),galactopinitol B (GPB), raffinose (Raf), stachyose (Sta), and sucrose(Suc).

FIGS. 18A-F are graphs showing accumulated soluble carbohydrates in axisand cotyledon tissues after precocious maturation of immature soybeanembryos as a function of time of slow drying. The results are shownafter feeding 100 mM D-chiro-inositol and 100 mM D-pinitol for 24 hoursat 25° C. followed by 0 to 14 days precocious maturation in slow dryingtime series relative humidities. Values are mean±SE (n=9).

FIGS. 18A-C are axis tissues. FIGS. 18D-F are cotyledon tissues.Abbreviations in FIGS. 18A-F are as follows: myo-inositol (myo),D-pinitol (Pin), D-chiro-inositol (chiro), fagopyritol B1 (B1),galactinol (Gol), galactopinitol A (GPA), galactopinitol B (GPB),raffinose (Raf), stachyose (Sta), and sucrose (Suc).

FIGS. 19A-B show GmGolS products. FIG. 19A shows galactinol (retentiontime 25.8 min) accumulation after enzyme incubation with 25 mMmyo-inositol, 25 mM UDP-Gal, 5 mM MnCl₂, and 2 mM DTT at 30° C. FIG. 19Bshows fagopyritol B1 (retention time 25.3 min) accumulation after enzymeincubation with 25 mM D-chiro-inositol, 25 mM UDP-Gal, 5 mM MnCl₂, and 2mM DTT at 30° C. Reactions were run to near completion to emphasizeproducts.

FIG. 20 shows pathways for biosynthesis of D-pinitol andD-chiro-inositol (from Obendorf, Seed Sci. Res. 7:63-74 (1997), which ishereby incorporated by reference in its entirety). The D-pinitolbiosynthetic pathway converts myo-inositol to D-ononitol to D-pinitol inlegume leaves. The illustration of D-pinitol on the bottom left isintentionally incorrectly numbered for clarity. 1L-myo-inositol6-O-methyltransferase (EC 2.1.1.129; also known as 1D-myo-inositol4-O-methyltransferase; reaction d) catalyzes the conversion ofmyo-inositol to D-ononitol. The conversion of D-ononitol to D-pinitol(e,f) may involve a two-step oxidoreductase reaction in soybean andother legumes: step 1, D-ononitol+NAD⁺→4-O-methyl-1D-myo-1-inosose+NADH;step 2, 4-O-methyl-1D-myo-1-inosose+NADPH→D-pinitol+NADP⁺. It isbelieved that D-chiro-inositol is formed by demethylation of D-pinitol(g,h), but neither the enzyme nor the gene have been identified.Prokaryotes, algae, insects, and animals appear to make D-chiro-inositolfrom myo-inositol (i,j). For details see Obendorf, Seed Sci. Res.7:63-74 (1997), which is hereby incorporated by reference in itsentirety. Earlier literature proposed that myo-inositol was converted toD-pinitol via sequoyitol (a,b,c) but the identity of sequoyitol is indoubt and may have been D-ononitol.

FIG. 21 shows the raffinose family oligosaccharides (RFO) and galactosylcyclitol biosynthetic pathways. GAS (or GolS), galactinol synthase; RFS,raffinose synthase; STS, stachyose synthase; VBS, verbascose synthase;GGT, galactan:galactan galactosyltransferase. Cyclitol may stand forD-ononitol, D-pinitol, or D-chiro-inositol, respectively. All reactionsare reversible (after Peterbauer et al., Seed Sci. Res. 11:185-198(2001), which is hereby incorporated by reference in its entirety).

FIG. 22 shows the revised RFO and galactosyl cyclitol biosyntheticpathways. GAS (or GolS), galactinol synthase; RFS, raffinose synthase;STS, stachyose synthase; VBS, verbascose synthase; GGT, galactan:galactan galactosyltransferase. All reactions are reversible (modifiedfrom Peterbauer et al., Seed Sci. Res. 11:185-198 (2001), which ishereby incorporated by reference in its entirety).

FIGS. 23A-C are graphs showing accumulation of major carbohydratesduring maturation of buckwheat embryos at 15, 22, and 30° C. Values(μg/embryo) are the mean±SE of the mean for three replicate samples.DAP=days after pollination.

FIGS. 24A-C are graphs showing the accumulation of D-chiro-inositol andits digalactosides, fagopyritol A2 and fagopyritol B2, during maturationof buckwheat embryos at 15, 22, and 30° C. Values (μg/embryo) are themean±SE of the mean for three replicate samples. DAP=days afterpollination.

FIGS. 25A-C are graphs showing the accumulation of myo-inositol and itsgalactosides, galactinol and digalactosyl myo-inositol, duringmaturation of buckwheat embryos at 15, 22, and 30° C. Values (μg/embryo)are the mean±SE of the mean for three replicate samples. DAP=days afterpollination.

FIGS. 26A-B are graphs showing seed germination rate (%) and seedlinghypocotyls length (mm) of buckwheat seeds matured at 15, 22, and 30° C.Values are the mean±SE of the mean for three replicate samples.

FIG. 27 shows the biosynthesis UDP-galactosamine fromα-D-galactose-1-phosphate and UDP-glucose usingUDP-glucose:α-D-galactose-1-phosphate uridylyltransferase (EC 2.7.7.9).

FIG. 28 shows biosynthesis of the putative insulin mediator(2-amino-2-deoxy-α-D-galactosamine-(1-3)-1D-chiro-inositol) and anisomer (2-amino-2-deoxy-α-D-galactosamine-(1-2)-1D-chiro-inositol) plusUDP, using UDP-galactosamine and D-chiro-inositol as substrates. Thereaction is catalyzed by the enzyme FeGolS-2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleic acid molecules encodingfagopyritol synthase enzymes. Fagopyritol is a general term used hereinto mean an unspecified α-galactosyl D-chiro-inositol or its salt orderivative. More particularly, the present invention relates to anisolated nucleic acid molecule encoding a fagopyritol synthase. Inaccordance with the present invention, the fagopyritol synthasecatalyzes the biosynthesis of a fagopyritol. Suitable fagopyritolsinclude fagopyritol A1, particularly fagopyritol A1s have the followingFormula I:

-   fagopyritol A2, particularly fagopyritol A2s having the following    Formula II:

-   fagopyritol A3, particularly fagopyritol A3s having the following    Formula III:

-   fagopyritol B1, particularly fagopyritol B1 s having the following    Formula IV:

-   fagopyritol B2, particularly fagopyritol B2s having the following    Formula V:

-   and fagopyritol B3, particularly fagopyritol B3s having the    following Formula VI:

One suitable source of a nucleic acid molecule encoding a fagopyritolsynthase enzyme is Fagopyrum esculentum.

In a first embodiment, the fagopyritol synthase from Fagopyrumesculentum is identified herein as FeGolS-1 and is encoded by a nucleicacid molecule having a nucleotide sequence of SEQ ID NO:1 as follows:

gagcacccaa agctctgcta gcaccatatt caaatcctca agaatcatca aatcttccaa   60ccaatcctca agttccaacc aaatggcacc agaactcatc acaatcggag ccgatcactc  120gattttgcca gcggaatcgt tgattccggt tgaccgagct tacgtgacgt ttctcgccgg  180gaacggagac tatgtcaagg gagttgtcgg attagcaaag ggactgagga aagtgaaggc  240tgcttatcct cttgttgtag cggttttacc ggacgttccg ctagagcatc gccgactcct  300ggaggcgcag ggttgtatcg taagggaaat cgagccgata tacccgccgg aaaacaattg  360cgagttcgct cacgcatact atgtcatcaa ctactccaag cttcgcatct gggagtttgt  420ggagtacagt aagatgatat acttggacgg ggacatacag gtgtaccaga acattgacca  480cctgtttgac cagccggacg gctactttta cgcggtgatg gactgttttt gtgagccatc  540atggagcaag acgattcagt acaagatcgg atactgccaa cagtgcccgg agaaggtagc  600gtggccgttg gaggctggcc cgaagccttc tctgtacttc aatgccggat tctttgttta  660cgagccgagc cttgagactt acaaggatct cattgacact ctcaaagtca cgactcctac  720ctcctttgcc gagcaggact tcttgaacat gtacttcaag gacaagttca agccactccc  780catagactac aacttagtct tagccttcct gtggaggcat ccggagaaag ttgaccttaa  840ccgagtgaag gtagttcact actgtgcggc ggggtctaag ccatggaggt acacgggcaa  900ggaagagaac atggacagag aagacatcaa attgcttgtg aaaaaatggt gggatatcta  960caacgacgag tcattggacc tcaagaaacc ggtccattta gtgcagcagc ccacggaggt 1020gctcaaggcg gcgctctcgg aggctaggcc tgttaaatat gtggctgctc cttccgcagc 1080ttaagtatcg gcttgtattt ggtaatggtt tttgtttttg cgaatgtaaa gtagaaagaa 1140ggggcgagag tttgtgatat tggggcaatg gggaatggtg cgtataaatg tgtgttgtaa 1200tggcaactgt ttttacttgg aattatatgt aagaagtaag aatatatgta taaaaaaaaa 1260aaaaaaaaa 1269

The nucleic acid sequence corresponding to SEQ ID NO:1 encodes anisoform of fagopyritol synthase isolated from Fagopyrum esculentum,identified herein as FeGolS-1, which has a deduced amino acid sequencecorresponding to SEQ ID NO:2, as follows:

Met Ala Pro Glu Leu Ile Thr Ile Gly Ala Asp His  1               5                  10        Ser Ile Leu Pro Ala Glu Ser Leu Ile Pro Val Asp         15                  20                Arg Ala Tyr Val Thr Phe Leu Ala Gly Asn Gly Asp 25                  30                  35Tyr Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu             40                  45Arg Lys Val Lys Ala Ala Tyr Pro Leu Val Val Ala     50                  55                  60Val Leu Pro Asp Val Pro Leu Glu His Arg Arg Leu                 65                  70Leu Glu Ala Gln Gly Cys Ile Val Arg Glu Ile Glu         75                  80Pro Ile Tyr Pro Pro Glu Asn Asn Cys Glu Phe Ala 85                  90                  95His Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg            100                 105Ile Trp Glu Phe Val Glu Tyr Ser Lys Met Ile Tyr    110                 115                 120Leu Asp Gly Asp Ile Gln Val Tyr Gln Asn Ile Asp                125                 130His Leu Phe Asp Gln Pro Asp Gly Tyr Phe Tyr Ala        135                 140Val Met Asp Cys Phe Cys Glu Pro Ser Trp Ser Lys145                 150                 155Thr Ile Gln Tyr Lys Ile Gly Tyr Cys Gln Gln Cys            160                 165Pro Glu Lys Val Ala Trp Pro Leu Glu Ala Gly Pro    170                 175                 180Lys Pro Ser Leu Tyr Phe Asn Ala Gly Phe Phe Val                185                 190Tyr Glu Pro Ser Leu Glu Thr Tyr Lys Asp Leu Ile        195                 200Asp Thr Leu Lys Val Thr Thr Pro Thr Ser Phe Ala205                 210                 215Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp Lys            220                 225Phe Lys Pro Leu Pro Ile Asp Tyr Asn Leu Val Leu    230                 235                 240Ala Phe Leu Trp Arg His Pro Glu Lys Val Asp Leu                245                 250Asn Arg Val Lys Val Val His Tyr Cys Ala Ala Gly        255                 260Ser Lys Pro Trp Arg Tyr Thr Gly Lys Glu Glu Asn265                 270                 275Met Asp Arg Glu Asp Ile Lys Leu Leu Val Lys Lys            280                 285Trp Trp Asp Ile Tyr Asn Asp Glu Ser Leu Asp Leu    290                 295                 300Lys Lys Pro Val His Leu Val Gln Gln Pro Thr Glu                305                 310Val Leu Lys Ala Ala Leu Ser Glu Ala Arg Pro Val        315                 320 Lys Tyr Val Ala Ala Pro Ser Ala Ala325                 330The fagopyritol synthase has a molecular mass of from 38 to 41 kDa, andpreferably 38.3 kDa. FeGoS-1, isolated from Fagopyrum esculentum(“buckwheat”), has a single open reading frame (“ORF”) of 1002 bp,extending between nucleotides 83-1084. The starting codon “ATG” isidentified at 83-85 bp, with the stop codon “TAA” found betweennucleotides 1082-1084, as shown in FIG. 1.

In a second embodiment, the fagopyritol synthase from Fagopyrumesculentum is identified herein as FeGolS-2 and is encoded by a nucleicacid molecule having a nucleotide sequence of SEQ ID NO:3 as follows:

ttggtttcga acttgatcaa aacctcacaa aaacacgtaa gcaaaatgac ttccgagatg   60gcgccacaga acataacgaa tgcagaaaga ggagccgagc aagtgaagcc gtcgagccag  120ccaagccgag cctacgtgac attcttagcc gggaacggtg actacgtgaa gggagttata  180gggctcgcca aaggcctgag gaaaactcag agcggttacc cgcttgtggt ggcggttctc  240cctgacgttc cgcaggagca ccgccgtatg ctggtggcgc aaggctgtat aataaaggaa  300atccagcccg ttaacccgcc cgataaccag actcagtttg ccatggctta ttacgtcatc  360aactactcca agctccgtat atgggagttt atcgagtata gtaagatgat atatcttgat  420ggagacatcc aagtttacga caacatcgac cacctcttcg acctaccaga cgggtacttg  480tacggtgcca tggattgctt ttgcgagaag acttggagtc attcgcttcc atataagatt  540gggtattgcc aacagtgccc ggacagggtc cagtggcccg aaaggctcgg cccaaaacca  600acactctact tcaatgcagg gatgttcatc ttcgagccta gcgtttctac ttataatgat  660ctccttcata cactcgagat cacccctcct acaccttttg ctgagcagga ctttttgaat  720atgtacttca aggatgtgta cagaccaatt ccgaacgttt acaacttggt attggctttg  780ttgtggtatc atcctgggtt aatgaagctt gatgaggtta aagtcgttca ctattgtgcc  840gatggttcaa aaccatggcg gtatacaggg aagggggata acatggacag ggaagacgtt  900aggatgctag tgaagaagtg gtgggagatt tacgatgatc agtctctcga ccctcagcct  960aagatggtcg agggcaagaa gttcgacaaa ttagaggagt acagcgagtc cctcgaccac 1020ccgcccaagg tggcagagga agataagcta gagaagccca tggcagcgat gacaggcttc 1080agctacgtac acgccccgtc tgctgcctga tttgttgaaa caaggccaag gttccacaaa 1140tgagggaatc aaaaacctcc tatagtatta tagatcgtat atttctgtta ttgctttcca 1200attaagcaac taagatgttc atatagtagt tctggaaaat gaatacgggc atagttgtga 1260acttgtaatc tcattttgtt tttcggaatg ttcaagtatt tcttctaaaa aaaaaaaaaa 1320aaaaaa 1326

The nucleic acid sequence corresponding to SEQ ID NO:3 encodes anisoform of fagopyritol synthase isolated from Fagopyrum esculentum,identified herein as FeGolS-2, which has a deduced amino acid sequencecorresponding to SEQ ID NO:4, as follows:

Met Thr Ser Glu Met Ala Pro Gln Asn Ile Thr Asn  1               5                  10Ala Glu Arg Gly Ala Glu Gln Val Lys Pro Ser Ser         15                  20Gln Pro Ser Arg Ala Tyr Val Thr Phe Leu Ala Gly 25                  30                  35Asn Gly Asp Tyr Val Lys Gly Val Ile Gly Leu Ala             40                  45Lys Gly Leu Arg Lys Thr Gln Ser Gly Tyr Pro Leu     50                  55                  60Val Val Ala Val Leu Pro Asp Val Pro Gln Glu His                 65                  70Arg Arg Met Leu Val Ala Gln Gly Cys Ile Ile Lys         75                  80Glu Ile Gln Pro Val Asn Pro Pro Asp Asn Gln Thr 85                  90                  95Gln Phe Ala Met Ala Tyr Tyr Val Ile Asn Tyr Ser            100                 105Lys Leu Arg Ile Trp Glu Phe Ile Glu Tyr Ser Lys    110                 115                 120Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Tyr Asp                125                 130Asn Ile Asp His Leu Phe Asp Leu Pro Asp Gly Tyr        135                 140Leu Tyr Gly Ala Met Asp Cys Phe Cys Glu Lys Thr145                 150                 155Trp Ser His Ser Leu Pro Tyr Lys Ile Gly Tyr Cys            160                 165Gln Gln Cys Pro Asp Arg Val Gln Trp Pro Glu Arg    170                 175                 180Leu Gly Pro Lys Pro Thr Leu Tyr Phe Asn Ala Gly                185                 190Met Phe Ile Phe Glu Pro Ser Val Ser Thr Tyr Asn        195                 200Asp Leu Leu His Thr Leu Glu Ile Thr Pro Pro Thr205                 210                 215Pro Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe            220                 225Lys Asp Val Tyr Arg Pro Ile Pro Asn Val Tyr Asn    230                 235                 240Leu Val Leu Ala Leu Leu Trp Tyr His Pro Gly Leu                245                 250Met Lys Leu Asp Glu Val Lys Val Val His Tyr Cys        255                 260Ala Asp Gly Ser Lys Pro Trp Arg Tyr Thr Gly Lys265                 270                 275Gly Asp Asn Met Asp Arg Glu Asp Val Arg Met Leu            280                 285Val Lys Lys Trp Trp Glu Ile Tyr Asp Asp Gln Ser    290                 295                 300Leu Asp Pro Gln Pro Lys Met Val Glu Gly Lys Lys                305                 310Phe Asp Lys Leu Glu Glu Tyr Ser Glu Ser Leu Asp        315                 320His Pro Pro Lys Val Ala Glu Glu Asp Lys Leu Glu325                 330                 335Lys Pro Met Ala Ala Met Thr Gly Phe Ser Tyr Val            340                 345 His Ala Pro Ser Ala Ala     350The fagopyritol synthase has a molecular mass of from 38 to 41 kDa, andpreferably 40.7 kDa. FeGoS-2, isolated from Fagopyrum esculentum, has asingle ORF of 1065 bp, extending between nucleotides 46-1110. Thestarting codon “ATG” is identified at 46-48 bp, with the stop codon“TGA” found between nucleotides 1108-1110, as shown in FIG. 2.

In a third embodiment, the fagopyritol synthase from Fagopyrumesculentum is identified herein as FeGolS-3 and comprises a nucleic acidmolecule having a nucleotide sequence of SEQ ID NO:5 (see FIG. 3) asfollows:

gctcacgcat actatgtcat caactactcc aagctccgta tatgggagtt tatcgagtat   60agtaagatga tatatcttga tggagacatc caagtttacg acaacatcga ccacctcttc  120gacctaccag acgggtactt gtacggtgcc atggattgct tttgcgagaa gacttggagt  180cattcgcttc catataagat tgggtattgc caacagtgcc cggacagggt ccagtggccc  240gaaaggctcg gcccaaaacc aacactctac ttcaatgcag ggatgttcat cttcgagcct  300agcgtttcta cttataatga tctccttcat acactcgaga tcacccctcc tacacctttt  360gctgagcagg actttttgaa tatgtacttc aaggatgtgt acagaccaat tccgaacgtg  420tacaacttgg tattggcttt gttgtggtat catcctgggt taatgaatct tgatgaggtt  480aaagtcgttc actattgtgc cgatggttca aaaccatggc ggtatacagg gaagggggat  540aacatggaca gggaagacgt taggatgcta gtgaagaagt ggtgggagat ctacgatgat  600cagtctctcg accctcagcc taaggtggtc gagggcaaga agttcgacaa attagagtac  660agcgagtccc tcgaccaccc gcctaaggtg gcagaggaag ataagttaga gaagcccatg  720gcggcgatga cagggttcag ctacgtacac gccccgtctg ctgcctgact tgttgaaaca  780aggccaaggt tccacaaatg agggaatcaa aaacctccta tagtattata gatcgtatat  840ttctgttatt gctttccaat taagcaacta agatgttcat atagtagttc tggaaaatga  900aaacgggcat agttgtgaac ttgtaatctc attttgtttt tcggaatgtg caagtatttc  960ttctaaataa aaaaaaaaaa aaaaaa  986

The nucleic acid sequence corresponding to SEQ ID NO:5 encodes anisoform of fagopyritol synthase isolated from Fagopyrum esculentum,identified herein as FeGolS-3, which comprises a deduced amino acidsequence corresponding to SEQ ID NO:6, as follows:

Ala His Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu  1               5                  10Arg Ile Trp Glu Phe Ile Glu Tyr Ser Lys Met Ile         15                  20Tyr Leu Asp Gly Asp Ile Gln Val Tyr Asp Asn Ile 25                  30                  35Asp His Leu Phe Asp Leu Pro Asp Gly Tyr Leu Tyr             40                  45Gly Ala Met Asp Cys Phe Cys Glu Lys Thr Trp Ser     50                  55                  60His Ser Leu Pro Tyr Lys Ile Gly Tyr Cys Gln Gln                 65                  70Cys Pro Asp Arg Val Gln Trp Pro Glu Arg Leu Gly         75                  80Pro Lys Pro Thr Leu Tyr Phe Asn Ala Gly Met Phe 85                  90                  95Ile Phe Glu Pro Ser Val Ser Thr Tyr Asn Asp Leu            100                 105Leu His Thr Leu Glu Ile Thr Pro Pro Thr Pro Phe    110                 115                 120Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp                125                 130Val Tyr Arg Pro Ile Pro Asn Val Tyr Asn Leu Val        135                 140Leu Ala Leu Leu Trp Tyr His Pro Gly Leu Met Asn145                 150                 155Leu Asp Glu Val Lys Val Val His Tyr Cys Ala Asp            160                 165Gly Ser Lys Pro Trp Arg Tyr Thr Gly Lys Gly Asp    170                 175                 180Asn Met Asp Arg Glu Asp Val Arg Met Leu Val Lys                185                 190Lys Trp Trp Glu Ile Tyr Asp Asp Gln Ser Leu Asp        195                 200Pro Gln Pro Lys Val Val Glu Gly Lys Lys Phe Asp205                 210                 215Lys Leu Glu Tyr Ser Glu Ser Leu Asp His Pro Pro            220                 225Lys Val Ala Glu Glu Asp Lys Leu Glu Lys Pro Met    230                 235                 240Ala Ala Met Thr Gly Phe Ser Tyr Val His Ala Pro                245                 250 Ser Ala Ala          255

Another suitable source of a nucleic acid molecule encoding afagopyritol synthase enzyme is Glycine max. A fagopyritol synthase fromGlycine max is identified herein as GmGolS and is encoded by a nucleicacid molecule having a nucleotide sequence of SEQ ID NO:7 as follows:

agccaaaagt ttgttttcat agtgtgtttt gtttcccaaa tcctactctt gtgaccacaa   60cccttcctcc tctttctttt gaaacctctt tttttctatt ccccaaccaa acaagcaaac  120gctactcact catcatcact gagatcatgg ctcctaatat caccactgtc aaaaccacca  180tcaccgacgc tcaagccaag gtcgccaccg atcatggtcg tgcctacgtc accttcctcg  240ccggaaacgg tgactatgtg aaaggtgtcg ttggcttggc aaaaggtctg agaaaagtga  300agagcatgta ccctctggtg gttgcagtgc tacccgatgt tccccaagat caccgcaaca  360ttctcacctc ccaaggttgc attgttagag agattgagcc cgtgtacccc ccagagaatc  420aaacccagtt tgccatggca tattacgtca tcaactattc caagctacgt atttgggagt  480ttgtggagta cagcaagatg atatacctag acggtgatat ccaagttttt gacaacattg  540accacttgtt tgacttgcct gataactact tctatgcggt gatggactgt ttctgtgagc  600caacttgggg ccacactaaa caatatcaga tcggttactg ccagcagtgc ccccataagg  660ttcagtggcc cactcacttt gggcccaaac ctcctctcta tttcaatgct ggcatgtttg  720tgtatgagcc caatttggct acttaccgtg acctccttca aacagtccaa gtcacccagc  780ccacttcctt tgctgaacag gattttttga acatttactt caaggacaaa tataggccaa  840ttcctaatgt ctacaatctt gtgctggcca tgctgtggcg tcaccctgag aacgttgagc  900ttgacaaagt taaagtggtt cactactgtg ctgctgggtc taagccttgg aggtacactg  960ggaaggagga gaatatggag agagaagata tcaagatgtt agtgaaaaag tggtgggata 1020tatatgagga tgagactttg gactacaaca atccactcaa tgtggataag ttcactgcgg 1080cacttatgga ggttggtgaa gtcaagttcg tccgtgcccc atctgctgct taagagtgtc 1140tttggaaatc aagtgtgatc caagtacatg tacaaagtca tacatcatta cattaacttt 1200tatgtatttc taaaagtcat acatcattac attaagtttt atgtatttct aaagtcttaa 1260gacttaagag gacctttttt atgtgtcccg gcttttcttt ttttcttttt ccaattctgt 1320cattgtaaag caggtgaata ccggtatcct taattttata aatggatatg aattttattt 1380tgcaaaaaaa aaaaaaaaaa aaaaaa 1406

The nucleic acid sequence corresponding to SEQ ID NO:7 encodes anisoform of fagopyritol synthase isolated from Glycine max, identifiedherein as GmGolS, which has a deduced amino acid sequence correspondingto SEQ ID NO:8, as follows:

Met Ala Pro Asn Ile Thr Thr Val Lys Thr Thr Ile  1               5                  10Thr Asp Ala Gln Ala Lys Val Ala Thr Asp His Gly         15                  20Arg Ala Tyr Val Thr Phe Leu Ala Gly Asn Gly Asp 25                  30                  35Tyr Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu             40                  45Arg Lys Val Lys Ser Met Tyr Pro Leu Val Val Ala     50                  55                  60Val Leu Pro Asp Val Pro Gln Asp His Arg Asn Ile                 65                  70Leu Thr Ser Gln Gly Cys Ile Val Arg Glu Ile Glu         75                  80Pro Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala 85                  90                  95Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg            100                 105Ile Trp Glu Phe Val Glu Tyr Ser Lys Met Ile Tyr    110                 115                 120Leu Asp Gly Asp Ile Gln Val Phe Asp Asn Ile Asp                125                 130His Leu Phe Asp Leu Pro Asp Asn Tyr Phe Tyr Ala        135                 140Val Met Asp Cys Phe Cys Glu Pro Thr Trp Gly His145                 150                 155Thr Lys Gln Tyr Gln Ile Gly Tyr Cys Gln Gln Cys            160                 165Pro His Lys Val Gln Trp Pro Thr His Phe Gly Pro    170                 175                 180Lys Pro Pro Leu Tyr Phe Asn Ala Gly Met Phe Val                185                 190Tyr Glu Pro Asn Leu Ala Thr Tyr Arg Asp Leu Leu        195                 200Gln Thr Val Gln Val Thr Gln Pro Thr Ser Phe Ala205                 210                 215Glu Gln Asp Phe Leu Asn Ile Tyr Phe Lys Asp Lys            220                 225Tyr Arg Pro Ile Pro Asn Val Tyr Asn Leu Val Leu    230                 235                 240Ala Met Leu Trp Arg His Pro Glu Asn Val Glu Leu                245                 250Asp Lys Val Lys Val Val His Tyr Cys Ala Ala Gly        255                 260Ser Lys Pro Trp Arg Tyr Thr Gly Lys Glu Glu Asn265                 270                 275Met Glu Arg Glu Asp Ile Lys Met Leu Val Lys Lys            280                 285Trp Trp Asp Ile Tyr Glu Asp Glu Thr Leu Asp Tyr    290                 295                 300Asn Asn Pro Leu Asn Val Asp Lys Phe Thr Ala Ala                305                 310Leu Met Glu Val Gly Glu Val Lys Phe Val Arg Ala        315                 320 Pro Ser Ala Ala 325(see FIG. 4). The fagopyritol synthase has a molecular mass ofapproximately 38.0 kDa.

Other suitable sources of nucleic acid molecules encoding fagopyritolsynthases include any plant that expresses galactinol synthase (i.e.,any plant that accumulates raffinose series of oligosaccharides),including, but not limited to, sugar beet, vetch, beans, legumes,cereals and grasses, cucurbits, and Brassicas (see, e.g., Kuo et al., J.Agricul. Food Chem. 36:32-36 (1988), which is hereby incorporated byreference in its entirety).

Fragments of the above fagopyritol synthase enzymes are encompassed bythe present invention.

Suitable fragments can be produced by several means. In one method,subclones of the genes encoding the fagopyritol synthase enzymes of thepresent invention are produced by conventional molecular geneticmanipulation by subcloning gene fragments. The subclones then areexpressed in vitro or in vivo in bacterial cells to yield a smallerprotein or peptide.

In another approach, based on knowledge of the primary structure of theprotein, fragments of a fagopyritol synthase enzyme encoding gene may besynthesized by using the PCR technique together with specific sets ofprimers chosen to represent particular portions of the protein. Thesethen would be cloned into an appropriate vector for increased expressionof a truncated peptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences for afagopyritol synthase enzyme being produced. Alternatively, subjecting afull length fagopyritol synthase enzyme to high temperatures andpressures will produce fragments. These fragments can then be separatedby conventional procedures (e.g., chromatography, SDS-PAGE).

Another example of suitable fragments of the nucleic acids of thepresent invention are fragments of the genes which have been identifiedas conserved (“con”) regions of the proteins, or alternatively, thoseportions of nucleotide sequences that have been identified as variable(“var”) regions. Sequences identified using DNAStar Mega alignmentprogram as either variable or conserved in a gene can be amplified usingstandard PCR methods using forward and reverse primers designed toamplify the region of choice and which include a restriction enzymesequence to allow ligation of the PCR product into a vector of choice.Combinations of amplified conserved and variable region sequences can beligated into a single vector to create a “cassette” which contains aplurality of DNA molecules in one vector.

Mutations or variants of the above polypeptides or proteins areencompassed by the present invention. Variants may be made by, forexample, the deletion or addition of amino acids that have minimalinfluence on the properties, secondary structure, and hydropathic natureof an enzyme. For example, a polypeptide may be conjugated to a signal(or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification, or identification of thepolypeptide.

Also suitable as an isolated nucleic acid molecule according to thepresent invention is a nucleic acid molecule having a nucleotidesequence that is at least 55% similar, preferably at least 80% similar,and most preferably, at least 90% similar, to the nucleotide sequence ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 by basic BLASTusing default parameters analysis.

Suitable nucleic acid molecules are those that hybridize to a nucleicacid molecule comprising a nucleotide sequence of SEQ ID No: 1, SEQ IDNO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 under stringent conditions. For thepurposes of defining the level of stringency, reference can convenientlybe made to Sambrook et al., Molecular Cloning: a Laboratory Manual,2^(nd) Edition, Cold Spring Harbor, N.Y., Cold Spring Harbor LaboratoryPress, at 11.45 (1989). An example of low stringency conditions is4-6×SSC/0.1-0.5% w/v SDS at 37°-45° C. for 2-3 hours. Depending on thesource and concentration of the nucleic acid involved in thehybridization, alternative conditions of stringency may be employed suchas medium stringent conditions. Examples of medium stringent conditionsinclude 1-4×SSC/0.25% w/v SDS at ≧45° C. for 2-3 hours. An example ofhigh stringency conditions includes 0.1-1×SSC/0.1% w/v SDS at 60° C. for1-3 hours. The skilled artisan is aware of various parameters which maybe altered during hybridization and washing and which will eithermaintain or change the stringency conditions. Other examples of highstringency conditions include: 4-5×SSC/0.1% w/v SDS at 54° C. for 1-3hours and 4×SSC at 65° C., followed by a washing in 0.1×SSC at 65° C.for about one hour. Alternatively, an exemplary stringent hybridizationcondition is in 50% formamide, 4×SSC, at 42° C. Still another example ofstringent conditions include hybridization at 62° C. in 6×SSC,0.05×BLOTTO, and washing at 2×SSC, 0.1% SDS at 62° C.

The precise conditions for any particular hybridization are left tothose skilled in the art because there are variables involved in nucleicacid hybridizations beyond those of the specific nucleic acid moleculesto be hybridized that affect the choice of hybridization conditions.These variables include: the substrate used for nucleic acidhybridization (e.g., charged vs. non-charged membrane); the detectionmethod used (e.g., radioactive vs. chemiluminescent); and the source andconcentration of the nucleic acid involved in the hybridization. All ofthese variables are routinely taken into account by those skilled in theart prior to undertaking a nucleic acid hybridization procedure.

A fagopyritol synthase enzyme of the present invention is preferablyproduced in purified form (e.g., at least about 80%, more preferably 90%pure) by conventional techniques. One example of a suitable technique isset forth in the Examples herein. Alternatively, a fagopyritol synthaseenzyme of the present invention is secreted into the growth medium ofrecombinant host cells. To isolate the fagopyritol synthase enzyme, aprotocol involving a host cell such as Escherichia coli may be used, inwhich protocol the E. coli host cell carrying a recombinant plasmid ispropagated, homogenized, and the homogenate is centrifuged to removebacterial debris. The supernatant is then subjected to sequentialammonium sulfate precipitation. The fraction containing the fagopyritolsynthase enzyme of the present invention is subjected to gel filtrationin an appropriately sized dextran or polyacrylamide column to separatethe proteins or polypeptides. If necessary, the protein fraction may befurther purified by high performance liquid chromatography (“HPLC”).

The nucleic acid molecule encoding the fagopyritol synthase enzyme ofthe present invention, or a suitable portion thereof, can beincorporated into host cells using conventional recombinant DNAtechnology. Generally, this involves inserting the nucleic acid moleculeinto an expression system to which the nucleic acid molecule isheterologous (i.e. not normally present). The expression system containsthe necessary elements for the transcription and translation of theinserted protein-coding sequences.

The present invention also relates to an expression vector containing anucleic acid molecule encoding a fagopyritol synthase enzyme of thepresent invention. The nucleic acid molecules of the present inventionmay be inserted into any of the many available expression vectors andcell systems using reagents that are well known in the art. In preparinga DNA vector for expression, the various DNA sequences may normally beinserted or substituted into a bacterial plasmid. Any convenient plasmidmay be employed, which will be characterized by having a bacterialreplication system, a marker which allows for selection in a bacterium,and generally one or more unique, conveniently located restrictionsites. Numerous plasmids, referred to as transformation vectors, areavailable for transformation. The selection of a vector will depend onthe preferred transformation technique and target cells fortransfection.

Suitable vectors include, but are not limited to, the following viralvectors such as lambda vector system gt11, gt WES.tB, Charon 4, andplasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/−or KS+/−(see “Stratagene Cloning Systems” Catalog (1993) fromStratagene, La Jolla, Calif., which is hereby incorporated by referencein its entirety), pQE, pIH821, pGEX, pET series (see F. W. Studier et.al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,”Gene Expression Technology vol. 185 (1990), which is hereby incorporatedby reference in its entirety), and any derivatives thereof. Anyappropriate vectors now known or later described for genetictransformation are suitable for use with the present invention.Recombinant molecules can be introduced into cells via transformation,particularly transduction, conjugation, mobilization, orelectroporation. The DNA sequences are cloned into the vector usingstandard cloning procedures in the art, as described by Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Press, NY (1989), and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., whichare hereby incorporated by reference in their entirety.

U.S. Pat. No. 4,237,224 issued to Cohen and Boyer, which is herebyincorporated by reference in its entirety, describes the production ofexpression systems in the form of recombinant plasmids using restrictionenzyme cleavage and ligation with DNA ligase. These recombinant plasmidsare then introduced by means of transformation and replicated inunicellular cultures including prokaryotic organisms and eukaryoticcells grown in tissue culture.

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria. Theexpression elements of these vectors vary in their strength andspecificities. Depending upon the host-vector system utilized, any oneof a number of suitable transcription and translation elements can beused.

Thus, certain “control elements” or “regulatory sequences” are alsoincorporated into the plasmid-vector constructs of the presentinvention. These include non-transcribed regions of the vector and 5′and 3′ untranslated regions, which interact with host cellular proteinsto carry out transcription and translation. Such elements may vary intheir strength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and/or translationelements, including constitutive, inducible, and repressible promoters,as well as minimal 5′ promoter elements may be used. A constitutivepromoter is a promoter that directs expression of a gene throughout thedevelopment and life of an organism. An inducible promoter is a promoterthat is capable of directly or indirectly activating transcription ofone or more DNA sequences or genes in response to an inducer. In theabsence of an inducer, the DNA sequences or genes will not betranscribed or will only be minimally transcribed.

The DNA sequences of eukaryotic promoters differ from those ofprokaryotic promoters. Furthermore, eukaryotic promoters andaccompanying genetic signals may not be recognized in or may notfunction in a prokaryotic system, and, further, prokaryotic promotersare not recognized and do not function in eukaryotic cells.

Promotors vary in their “strength” (i.e. their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promotors in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promotors maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promotors such as the T7 phage promoter, lac promotor, trppromotor, recA promotor, ribosomal RNA promotor, the P_(R) and P_(L)promotors of coliphage lambda and others, including but not limited, tolacUV5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promotor or other E. coli promotors produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Other examples of some constitutive promoters that are widely used forinducing expression of transgenes include the nopoline synthase (NOS)gene promoter, from Agrobacterium tumefaciens, (U.S. Pat. No. 5,034,322issued to Rogers et al., which is hereby incorporated by reference inits entirety), the cauliflower mosaic virus (CaMV) 35S and 19S promoters(U.S. Pat. No. 5,352,605 issued to Fraley et al., which is herebyincorporated by reference in its entirety), the enhanced CaMV35Spromoter (“enh CaMV35S”), the figwort mosaic virus full-lengthtranscript promoter (“FMV35S”), those derived from any of the severalactin genes, which are known to be expressed in most cells types (U.S.Pat. No. 6,002,068 issued to Privalle et al., which is herebyincorporated by reference in its entirety), and the ubiquitin promoter,which is a gene product known to accumulate in many cell types. Examplesof constitutive promoters for use in mammalian cells include the RSVpromoter derived from Rous sarcoma virus, the CMV promoter derived fromcytomegalovirus, β-actin and other actin promoters, and the EF1αpromoter derived from the cellular elongation factor 1α gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promoter unless specifically induced. Incertain operations, the addition of specific inducers is necessary forefficient transcription of the inserted nucleic acid. For example, thelac operon is induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Other examples of some inducible promoters, induced, for examples by achemical agent, such as a metabolite, growth regulator, herbicide orphenolic compound, or a physiological stress/physical means, such ascold, heat, salt, toxins, or through the action of a pathogen or diseaseagent such as a virus or fungus, include a glucocorticoid-induciblepromoter (Schena et al., Proc. Natl. Acad. Sci. 88:10421-5 (1991), whichis hereby incorporated by reference in its entirety), the heat shockpromoter (“Hsp”), IPTG or tetracycline (“Tet on” system), themetallothionine promoter, which is activated by heavy metal ions, andhormone-responsive promoters, which are activated by treatment ofcertain hormones. A host cell containing an inducible promoter may beexposed to an inducer by externally applying the inducer to the cell. Inaddition, “tissue-specific” promoters can be used, which are promotersthat function in a tissue specific manner to regulate the gene ofinterest within selected tissues of the host. Examples of such tissuespecific promoters include seed, flower, or root specific promoters asare well known in the field (e.g., U.S. Pat. No. 5,750,385 to Shewmakeret al., which is hereby incorporated by reference in its entirety).Promoters of the nucleic acid construct of the present invention may beeither homologous (derived from the same species as the host cell) orheterologous (derived from a different species than the host cell.

Specific initiation signals are also required for efficient genetranscription and translation in prokaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires an SD sequence about 7-9 bases 5′ to the initiationcodon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATGcombination that can be utilized by host cell ribosomes may be employed.Such combinations include but are not limited to the SD-ATG combinationfrom the cro gene or the N gene of coliphage lambda, or from the E. colitryptophan E, D, C, B or A genes. Additionally, any SD-ATG combinationproduced by recombinant DNA or other techniques involving incorporationof synthetic nucleotides may be used.

The constructs of the present invention also include an operable 3′regulatory region, selected from among those which are capable ofproviding correct transcription termination and polyadenylation of mRNAfor expression in the host cell of choice, operably linked to a DNAmolecule which encodes for a protein of choice. A number of 3′regulatory regions are known in the art. Virtually any 3′ regulatoryregion known to be operable in the host cell of choice would suffice forproper expression of the coding sequence of the nucleic acid of thepresent invention.

In one aspect of the present invention, the nucleic acid molecule of thepresent invention is incorporated into an appropriate vector in thesense direction, such that the open reading frame is properly orientedfor the expression of the encoded protein under control of a promoter ofchoice. This involves the inclusion of the appropriate regulatoryelements into the DNA-vector construct. These include non-translatedregions of the vector, useful promoters, and 5′ and 3′ untranslatedregions which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used.

A nucleic acid molecule of the preset invention, promoter of choice, anappropriate 3′ regulatory region, and, if desired, a reporter gene, canbe incorporated into a vector-expression system which contains thenucleic acids of the present invention, or suitable fragments thereof,using standard cloning techniques as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Press, NY (1989), and Ausubel et al. (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y., which are herebyincorporated by reference in their entirety. The transcriptional andtranslational elements are operably linked to the nucleic acid moleculeof the present invention or a fragment thereof, meaning that theresulting vector expresses the fagopyritol synthase when placed in asuitable host cell.

Once an isolated DNA molecule encoding a fagopyritol synthase enzyme hasbeen cloned into an expression vector, it is ready to be incorporatedinto a host cell. Such incorporation can be carried out by the variousforms of transformation noted above, depending upon the vector/host cellsystem. Recombinant molecules can be introduced into cells viatransformation, particularly transduction, conjugation, mobilization, orelectroporation. The nucleic acid sequences are cloned into the hostcell using standard cloning procedures known in the art, as described bySambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which ishereby incorporated by reference in its entirety. Suitable host cellsinclude, but are not limited to, bacteria, virus, yeast, mammaliancells, insect, plant, and the like.

Thus, the present invention also relates to a host cell incorporatingone or more of the isolated nucleic acid molecules of the presentinvention. In one embodiment, the isolated nucleic acid molecule isheterologous to the host cell. Such incorporation can be carried out bythe various forms of transformation noted above, depending upon thevector/host system, and using the various host cells described above.

Methods of transformation may result in transient or stable expressionof the DNA under control of the promoter. Preferably, the nucleic acidof the present invention is stably inserted into the genome of the hostcell as a result of the transformation, although transient expressioncan serve an important purpose.

One approach to transforming host cells with a nucleic acid molecule ofthe present invention is particle bombardment (also known as biolistictransformation) of the host cell. This can be accomplished in one ofseveral ways. The first involves propelling inert or biologically activeparticles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which arehereby incorporated by reference in their entirety. Generally, thisprocedure involves propelling inert or biologically active particles atthe cells under conditions effective to penetrate the outer surface ofthe cell and to be incorporated within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.Other variations of particle bombardment, now known or hereafterdeveloped, can also be used.

Transient expression in protoplasts allows quantitative studies of geneexpression, because the population of cells is very high (on the orderof 10⁶). To deliver DNA inside protoplasts, several methodologies havebeen proposed, but the most common are electroporation (Fromm et al.,Proc. Natl. Acad. Sci. USA 82:5824-5828 (1985), which is herebyincorporated by reference in its entirety) and polyethylene glycol (PEG)mediated DNA uptake (Krens et al., Nature 296:72-74 (1982), which ishereby incorporated by reference in its entirety). Duringelectroporation, the DNA is introduced into the cell by means of areversible change in the permeability of the cell membrane due toexposure to an electric field. PEG transformation introduces the DNA bychanging the elasticity of the membranes. Unlike electroporation, PEGtransformation does not require any special equipment and transformationefficiencies can be equally high. Another appropriate method ofintroducing the nucleic acid molecule of the present invention into ahost cell is fusion of protoplasts with other entities, eitherminicells, cells, lysosomes, or other fusible lipid-surfaced bodies thatcontain the chimeric gene (Fraley, et al., Proc. Natl. Acad. Sci. USA76:3348-52 (1979), which is hereby incorporated by reference in itsentirety).

Stable transformants are preferable for the methods of the presentinvention. An appropriate method of stably introducing the nucleic acidmolecule into plant cells is to infect a plant cell with Agrobacteriumtumefaciens or Agrobacterium rhizogenes previously transformed with aDNA construct of the present invention. Under appropriate conditionsknown in the art, the transformed plant cells are grown to form shootsor roots, and develop further into plants.

Plant tissues suitable for transformation include without limitation,floral buds, leaf tissue, root tissue, meristems, zygotic and somaticembryos, megaspores, callus, protoplasts, tassels, pollen, embryos,anthers, and the like. The means of transformation chosen is that mostsuited to the tissue to be transformed.

Suitable plants include dicots and monocots. Monocots suitable for thepresent invention include Gramineae (e.g., grass, corn, grains, bamboo,sugar cane), Liliaceae (e.g., onion, garlic, asparagus, tulips,hyacinths, day lily, and aloes), Iridaceae (e.g., iris, gladioli,freesia, crocus, and watsonia), and Orchidacea (e.g., orchid). Examplesof dicots suitable for the present invention include Salicaceae (e.g.,willow, and poplar), Ranunculaceae (e.g., Delphinium, Paeonia,Ranunculus, Anemone, Clematis, columbine, and marsh marigold),Magnoliaceae (e.g., tulip tree and Magnolia), Cruciferae (e.g.,mustards, cabbage, cauliflower, broccoli, brussel sprouts, kale,kohlrabi, turnip, and radish), Rosaceae (e.g., strawberry, blackberry,peach, apple, pear, quince, cherry, almond, plum, apricot, and rose),Leguminosae (e.g., pea, bean, peanut, alfalfa, clover, vetch, redbud,broom, wisteria, lupine, black locust, and acacia), Malvaceae (e.g.,cotton, okra, and mallow), Umbelliferae (e.g., carrot, parsley,parsnips, and hemlock), Labiatae (e.g., mint, peppermints, spearmint,thyme, sage, and lavender), Solanaceae (e.g., potato, tomato, pepper,eggplant, and Petunia), Cucurbitaceae (e.g., melon, squash, pumpkin, andcucumber), Compositae (e.g., sunflower, endive, artichoke, lettuce,safflower, aster, marigold, dandelions, sage brush, Dalia,Chrysanthemum, and Zinna), and Rubiaceae (e.g., coffee).

After transformation, the transformed plant cells can be selected andregenerated. Preferably, transformed cells are first identified using aselection marker simultaneously introduced into the host cells alongwith the DNA construct of the present invention. Suitable selectionmarkers include, without limitation, markers encoding for antibioticresistance, such as the nptII gene which confers kanamycin resistance(Fraley, et al., Proc. Natl. Acad. Sci. USA 80:4803-4807 (1983), whichis hereby incorporated by reference in its entirety), and the geneswhich confer resistance to gentamycin, G418, hygromycin, streptomycin,spectinomycin, tetracycline, chloramphenicol, and the like. Any knownantibiotic-resistance marker can be used to transform and selecttransformed host cells in accordance with the present invention. Cellsor tissues are grown on a selection medium containing the appropriateantibiotic, whereby generally only those transformants expressing theantibiotic resistance marker continue to grow. Other types of markersare also suitable for inclusion in the expression cassette of thepresent invention. For example, a gene encoding for herbicide tolerance,such as tolerance to sulfonylurea is useful, or the dhfr gene, whichconfers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104(1983), which is hereby incorporated by reference in its entirety).Similarly, “reporter genes,” which encode for enzymes providing forproduction of a compound identifiable are suitable. The most widely usedreporter gene for gene fusion experiments has been uidA, a gene fromEscherichia coli that encodes the β-glucuronidase protein, also known asGUS (Jefferson et al., EMBO J. 6:3901-3907 (1987), which is herebyincorporated by reference in its entirety). Similarly, enzymes providingfor production of a compound identifiable by luminescence, such asluciferase, are useful. The selection marker employed will depend on thetarget species; for certain target species, different antibiotics,herbicide, or biosynthesis selection markers are preferred.

Once a recombinant plant cell or tissue has been obtained, it ispossible to regenerate a full-grown plant therefrom. It is known thatpractically all plants can be regenerated from cultured cells ortissues, including but not limited to, all major species of sugarcane,sugar beets, cotton, fruit trees, and legumes. Means for regenerationvary from species to species of plants, but generally a suspension oftransformed protoplasts or a petri plate containing transformed explantsis first provided. Callus tissue is formed and shoots may be inducedfrom callus and subsequently rooted. Alternatively, embryo formation canbe induced in the callus tissue. These embryos germinate as naturalembryos to form plants. The culture media will generally contain variousamino acids and hormones, such as auxin and cytokinins. It is alsoadvantageous to add glutamic acid and proline to the medium, especiallyfor such species as corn and alfalfa. Efficient regeneration will dependon the medium, on the genotype, and on the history of the culture. Ifthese three variables are controlled, then regeneration is usuallyreproducible and repeatable.

Plant regeneration from cultured protoplasts is described in Evans, etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III(1986), which are hereby incorporated by reference in their entirety.

After the DNA construct is stably incorporated in transgenic plants, itcan be transferred to other plants by sexual crossing or by preparingcultivars. With respect to sexual crossing, any of a number of standardbreeding techniques can be used depending upon the species to becrossed. Cultivars can be propagated in accord with common agriculturalprocedures known to those in the field. Alternatively, transgenic seedsor propagules (e.g., cuttings) are recovered from the transgenic plants.The seeds can then be planted in the soil and cultivated usingconventional procedures to produce transgenic plants.

Another aspect of the present invention relates to a method forproducing a fagopyritol, an insulin mediator, an insulin mediatoranalogue, an insulin mediator homologue, or an insulin mediatorinhibitor. As used herein, fagopyritols, insulin mediators, insulinmediator analogues, insulin mediator homologues, and insulin mediatorinhibitors include salts and derivatives thereof.

Studies have been completed that link Type II diabetes and PCOS todeficiencies in insulin mediators composed of galactosamineD-chiro-inositol. Although their functions have yet to be fullycharacterized, it is known that insulin mediators act as secondmessengers of insulin action, and they are believed to be inositolphosphoglycans bound to cell membranes (Lamer et al., Diabetes Reviews7:217-231 (1999), which is hereby incorporated by reference in itsentirety). In the presence of insulin, these mediators are released andmay activate glycogen synthesis. It has been found that feedingD-chiro-inositol to women with PCOS increased insulin response andovulatory function (Nestler et al., N. Engl. J. Med. 340:1314-1320(1999), which is hereby incorporated by reference in its entirety).Another study has also shown that insulin resistance has been associatedwith abnormal D-chiro-inositol metabolism (Ortmeyer et al.,Endocrinology 132:640-645 (1993), which is hereby incorporated byreference in its entirety). Thus, synthesis of insulin mediatorscontaining D-chiro-inositol is of importance in order to determine atreatment for Type II diabetes and PCOS.

This method of the present invention includes providing a fagopyritolsynthase, providing a substrate including a galactosyl donor and agalactosyl acceptor, and combining the fagopyritol synthase with thesubstrate under conditions effective to produce a fagopyritol, aninsulin mediator, an insulin mediator analogue, an insulin mediatorhomologue, or an insulin mediator inhibitor.

Suitable fagopyritols which can be produced by the above method of thepresent invention are described above.

Suitable insulin mediators, insulin mediator analogues, insulin mediatorhomologues, and insulin mediator inhibitors which can be produced by theabove method of the present invention include, but are not limited to,galactosamine-D-chiro-inositols, galactosamine L-chino-inositols,galactosamine-myo-inositols, galactosamine-scyllo-inositols,galactosamine-bornesitols, galactose-D-chiro-inositols, galactoseL-chiro-inositols, galactose-myo-inositols, galactose-scyllo-inositols,galactose-bornesitols, glucose-D-chino-inositols, glucoseL-chino-inositols, glucose-myo-inositols, glucose-scyllo-inositols,glucose-bornesitols, glucosamine-D-chino-inositols, glucosamineL-chiro-inositols, glucosamine-myo-inositols,glucosamine-scyllo-inositols, and glucoseamine-bornesitols.

Suitable galactosyl donors include, but are not limited to,UDP-galactose, UDP-galactosamine, UDP-glucose, and UDP-glucosamine,which may be used with the enzymes described herein or enzyme mutants.

Suitable galactosyl acceptors include, but are not limited to,D-chiro-inositol, L-chiro-inositol, myo-inositol, bornesitol, andscyllo-inositol.

The fagopyritol synthase and substrate are combined to produce afagopyritol, an insulin mediator, an insulin mediator analogue, or aninsulin mediator homologue. Suitable conditions are determined by thefagopyritol synthase and substrate used, and include suitable amounts ofMn²⁺ (e.g., approximately 1-15 mM MnCl₂, preferably 5 mM MnCl₂) andsuitable amounts of reducing agents, such as DTT and mercaptoethanol.One example of suitable conditions is disclosed in the enzyme assaysdescribed in the Examples, below.

Separation of the resulting fagopyritol, insulin mediator, insulinmediator analogue, or insulin mediator homologue from any othercomponents may be achieved by methods known to one of ordinary skill inthe art, such as with carbon-Celite, BioRad P2 gel, TLC, HPLC, or Dowexcolumns.

Thus, the method of the present invention can be used to produce anisolated or substantially pure fagopyritol, insulin mediator, insulinmediator analogue, insulin mediator homologue, insulin mediatorinhibitor, or salts or derivatives thereof. As used herein, an isolatedfagopyritol, insulin mediator, insulin mediator analogue, insulinmediator homologue, or insulin mediator inhibitor, is one which issubstantially free of other components with which it naturally occurs.As referred to herein, substantially pure means substantially free ofother compounds or materials, such as galactinol, myo-inositol,digalactosyl myo-inositol, phytin, aromatic materials (e.g. polyphenolsand pigments and other colored aromatic materials), cell wall particles,proteins, and acids (e.g. organic acids, nucleic acids, and amino acids)and their salts. Typically, substantially pure fagopyritols, insulinmediators, insulin mediator analogues, insulin mediator homologues, orinsulin mediator inhibitors are those having greater than about 95%purity, such as greater than about 98% purity or from about 95% to about99% purity.

Salts of the fagopyritols can be the reaction product of a base having apKa (i.e., −log Ka) greater than the pKa of one or more of thefagopyritols' hydroxyl groups, such as a metal hydroxide or alkoxide, anamonium hydroxide, or an amine (e.g. a tertiary amine, like triethylamine). Exemplary salts are alkali metal salts, such as lithium salts,sodium salts, and potassium salts, alkali earth metal salts, such ascalcium salts and barium salts, ammonium salts, sufonium salts, andphosphonium salts.

Derivatives of the fagopyritols, include, for example, the reactionproducts of the fagopyritols with compounds bearing a carbon having apositive charge, such as an alkyl halide, in which case the derivativeis an ether of the fagopyritol, or a carboxylic acid halide (e.g.,acetyl chloride) or anhydride (e.g., acetic anhydride), in which casethe derivative is an ester of the fagopyritol (e.g., the acetate).

The fagopyritols, insulin mediators, insulin mediator analogues, insulinmediator homologues, and insulin mediator inhibitors produced with thefagopyritol synthase genes of the present invention can be used in acomposition which includes one or more of fagopyritol A1, fagopyritolA2, fagopyritol A3, fagopyritol B1, fagopyritol B2, fagopyritol B3,D-chiro-inositol, an insulin mediator, an insulin mediator analogue, aninsulin mediator homologue, or an insulin mediator inhibitor.Preferably, the composition is substantially free of one or more ofgalactinol, myo-inositol, digalactosyl myo-inositol, phytin, aromaticmaterials (e.g. polyphenols and pigments and other colored aromaticmaterials), cell wall particles, proteins, and acids (e.g. organicacids, nucleic acids, and amino acids) and their salts. It was observedthat a mixture of fagopyritols was degraded within six hours in thepresence of human fecal bacteria under in vitro conditions in thelaboratory. Therefore, it is believed that the fagopyritols are digestedby bacteria in the digestive tract to release free D-chiro-inositol foruptake, or in the case of monomers or dimers, may be taken up by cellsof the digestive tract.

The aforementioned fagopyritols, insulin mediators, insulin mediatoranalogues, insulin mediator homologues, insulin mediator inhibitors, andcompositions are useful in treating diabetes in patients, such asmammals, including dogs, cats, rats, mice, and humans, by administeringan effective amount of isolated or substantially pure fagopyritols,insulin mediators, insulin mediator analogues, insulin mediatorhomologues, insulin mediator inhibitors, or compositions to suchpatients. The aforementioned fagopyritols, insulin mediators, insulinmediator analogues, insulin mediator homologues, insulin mediatorinhibitors, and compositions may also be useful in treating polycysticovary syndrome (see Nestler et al., New England J. of Med.,340:1314-1320 (1999), which is hereby incorporated by reference in itsentirety). For example, the substantially pure fagopyritols, insulinmediators, insulin mediator analogues, insulin mediator homologues, andinsulin mediator inhibitors, the compositions, or one or more isolatedfagopyritols, insulin mediators, insulin mediator analogues, insulinmediator homologues, and insulin mediator inhibitors can be administeredalone, or in combination with suitable pharmaceutical carriers ordiluents. The diluent or carrier ingredients should be selected so thatthey do not diminish the therapeutic effects of the fagopyritols,insulin mediators, insulin mediator analogues, insulin mediatorhomologues, insulin mediator inhibitors, or compositions. Suitablepharmaceutical compositions include those which include a pharmaceuticalcarrier and, for example, one or more of an isolated fagopyritol A1, anisolated fagopyritol A2, an isolated fagopyritol A3, an isolatedfagopyritol B1, an isolated fagopyritol B2, an isolated fagopyritol B3,an insulin mediator, an insulin mediator analogue, an insulin mediatorhomologue, or an insulin mediator inhibitor.

The fagopyritols, insulin mediators, insulin mediator analogues, insulinmediator homologues, insulin mediator inhibitors, and compositionsherein can be made up in any suitable form appropriate for the desireduse; e.g., oral, parenteral, or topical administration. Examples ofparenteral administration are intraventricular, intracerebral,intramuscular, intravenous, intraperitoneal, rectal, and subcutaneousadministration. The preferred route for administration is oral. In caseswhere the fagopyritols, insulin mediators, insulin mediator analogues,insulin mediator homologues, or insulin mediator inhibitors, areadministered topically or parenterally, it is preferred that they bepre-hydrolyzed.

Suitable dosage forms for oral use include tablets, dispersible powders,granules, capsules, suspensions, syrups, and elixirs. Inert diluents andcarriers for tablets include, for example, calcium carbonate, sodiumcarbonate, lactose, and talc. Tablets may also contain granulating anddisintegrating agents, such as starch and alginic acid; binding agents,such as starch, gelatin, and acacia; and lubricating agents, such asmagnesium stearate, stearic acid, and talc. Tablets may be uncoated ormay be coated by known techniques to delay disintegration andabsorption. Inert diluents and carriers which may be used in capsulesinclude, for example, calcium carbonate, calcium phosphate, and kaolin.Suspensions, syrups, and elixirs may contain conventional excipients,such as methyl cellulose, tragacanth, sodium alginate; wetting agents,such as lecithin and polyoxyethylene stearate; and preservatives, suchas ethyl-p-hydroxybenzoate. Dosage forms suitable for parenteraladministration include solutions, suspensions, dispersions, emulsions,and the like. They may also be manufactured in the form of sterile solidcompositions which can be dissolved or suspended in sterile injectablemedium immediately before use. They may contain suspending or dispersingagents known in the art.

For oral administration either solid or fluid unit dosage forms can beprepared. For preparing solid compositions, such as tablets, a suitablefagopyritol, insulin mediator, insulin mediator analogue, insulinmediator homologue, insulin mediator inhibitor, or composition, asdisclosed above, is mixed with conventional ingredients, such as talc,magnesium stearate, dicalcium phosphate, magnesium aluminum silicate,calcium sulfate, starch, lactose, acacia methylcellulose, andfunctionally similar materials as pharmaceutical diluents or carriers.Capsules are prepared by mixing the disclosed fagopyritols, insulinmediators, insulin mediator analogues, insulin mediator homologues,insulin mediator inhibitors, or compositions with an inertpharmaceutical diluent and filling the fixture into a hard gelatincapsule of appropriate size. Soft gelatin capsules are prepared bymachine encapsulation of a slurry of the fagopyritol, insulin mediator,insulin mediator analogue, insulin mediator homologue, insulin mediatorinhibitor, or composition with an acceptable vegetable oil, light liquidpetrolatum, or other inert oil.

Fluid unit dosage forms for oral administration such as syrups, elixirs,and suspensions can be prepared. The water-soluble forms can bedissolved in an aqueous vehicle together with sugar, aromatic flavoringagents, and preservatives to form a syrup. An elixir is prepared byusing a hydro-alcoholic (ethanol) vehicle with suitable sweeteners, suchas sugar and saccharin, together with an aromatic flavoring agent.Suspensions can be prepared with a syrup vehicle with the aid of asuspending agent, such as acacia, tragacanth, methylcellulose, and thelike.

When the fagopyritols, insulin mediators, insulin mediator analogues,insulin mediator homologues, insulin mediator inhibitors, orcompositions are administered orally, suitable daily dosages can bebased on suitable doses of free D-chiro-inositol, such as thosedescribed in U.S. Pat. No. 5,124,360 to Lamer et al., which is herebyincorporated by reference in its entirety. It is believed that abouthalf of the fagopyritols as extracted is D-chiro-inositol, mostly asbound D-chiro-inositol with small amounts of free D-chiro-inositol.Therefore, suitable doses of fagopyritol are about twice the suitabledoses of D-chiro-inositol. Typically, for oral administration, suitabledaily doses are from about 5 mg to about 200 mg of the fagopyritol orcomposition per kilogram of the subject's body weight.

Alternatively, the fagopyritols, insulin mediators, insulin mediatoranalogues, insulin mediator homologues, or insulin mediator inhibitors,can be administered orally in foodstuffs. For example, fagopyritols canbe incorporated in purified form or in the form of buckwheat bran inbread, bread rolls, or other foodstuffs to form an edible product forconsumption of fagopyritols. Fortification of breads, bread rolls, andother foodstuffs with synthesized fagopyritols, insulin mediators,insulin mediator analogues, insulin mediator homologues, or insulinmediator inhibitors can provide a way to incorporate larger quantitiesof fagopyritols, insulin mediators, insulin mediator analogues, insulinmediator homologues, or insulin mediator inhibitors into a daily diet.Suitable procedures for bread preparation can be found, for example, inBrown, The Tassajara Bread Book, Boston: Shambhala Publications (1986),which is hereby incorporated by reference.

For parenteral administration, fluid unit dosage forms are preparedutilizing the aforementioned fagopyritols, insulin mediators, insulinmediator analogues, insulin mediator homologues, insulin mediatorinhibitors, or compositions and a sterile vehicle, water beingpreferred. The fagopyritol, insulin mediator, insulin mediator analogue,insulin mediator homologue, insulin mediator inhibitor, or composition,depending on the vehicle and concentration used, can be either suspendedor dissolved in the vehicle. In preparing solutions, the fagopyritol,insulin mediator, insulin mediator analogue, insulin mediator homologue,insulin mediator inhibitor, or composition can be dissolved in water forinjection and filter sterilized before filling into a suitable vial orampule and sealing. Advantageously, adjuvants, such as a localanesthetic, preservative, and buffering agents, can be dissolved in thevehicle. To enhance the stability, the fluid unit dosage form can befrozen after filling into the vial, and the water removed under vacuum.The dry lyophilized powder is then sealed in the vial, and anaccompanying vial of water for injection is supplied to reconstitute theliquid prior to use. Parenteral suspensions are prepared insubstantially the same manner except that the fagopyritol, insulinmediator, insulin mediator analogue, insulin mediator homologue, insulinmediator inhibitor, or composition is suspended in the vehicle insteadof being dissolved, and sterilization cannot be accomplished byfiltration. The fagopyritol, insulin mediator, insulin mediatoranalogue, insulin mediator homologue, insulin mediator inhibitor, orcomposition can be sterilized by exposure to ethylene oxide beforesuspending in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in the parenteral suspension to facilitateuniform distribution of the fagopyritol, insulin mediator, insulinmediator analogue, insulin mediator homologue, insulin mediatorinhibitor, or composition. Parenteral dosages can range from about 5 mgto about 200 mg of fagopyritol, insulin mediator, insulin mediatoranalogue, insulin mediator homologue, insulin mediator inhibitor, orcomposition per kilogram of the subject's body weight per day.Preferably, the daily parenteral dosage would be considerably less thanthe dose per kilogram of subject body weight, considering that, in oraladministration, the galactose from the fagopyritols would be consumed bymicrobes in the digestive tract whereas, in parenteral administrationthe galactose would contribute to blood sugar levels.

Alternatively, the fagopyritol, insulin mediator, insulin mediatoranalogue, insulin mediator homologue, insulin mediator inhibitor, orcomposition can be incorporated into a sustained release formulation andsurgically implanted using conventional methods. Suitable sustainedrelease matricies include those made of ethylene vinyl acetate and otherbicompatible polymers.

For topical administration, carriers, such as phospholipid vesicles,which contain the aforementioned fagopyritols, insulin mediators,insulin mediator analogues, insulin mediator homologues, or insulinmediator inhibitors, may facilitate uptake through the skin.

As indicated above, it is believed that the fagopyritols are digested inthe digestive tract by bacteria to release free D-chiro-inositol foruptake. It is known that D-chiro-inositol is an anti-oxidant and, moreparticularly, a hydroxyl radical scavenger. Accordingly, the fagopyritoland compositions can also be used as a source of the antioxidantD-chiro-inositol, for example, by administering, preferably orally, thesubject fagopyritols and compositions to a subject.

The present invention is further illustrated by the following examples.

EXAMPLES Example 1 Fagopyritol Synthase, A Novel Multi-FunctionalGalactinol Synthase Homologue, Catalyzes the Biosynthesis of FagopyritolA1 and Fagopyritol B1 in Buckwheat Seeds

Nucleotide and Amino Acid Sequence Analyses

The nucleotide sequences of galactinol synthase genes identified to dateand their corresponding amino acid sequences were obtained from thenucleotide and protein databases (http://www.ncbi.nlm.nih.gov).Nucleotide and amino acid sequences were compared using a multiplesequence alignment program, CLUSTAL W (http://workbench.sdsc.edu). Theidentities of buckwheat cDNA fragments amplified from RT-PCR andRACE-PCR assays were examined by BLASTN and BLASTX programs(http://www.ncbi.nlm.nih.gov and http://workbench.sdsc.edu).

Isolation of FeGolS cDNA

The synthesis of PCR-directed cDNA from the poly(A)⁺RNA isolated fromdeveloping buckwheat seeds (harvested at 20 to 25 days afterpollination) was described previously (Lewis et al., Gene 246:81-91(2000), which is hereby incorporated by reference in its entirety).Briefly, it involved the synthesis of the first strand cDNA using anoligo-dT primer (primer A, 5′-GCGGCCGCTTTTTTTTTTTTTTTTT-3′ (SEQ IDNO:16), FIG. 5) and reverse transcriptase, followed byoligo-dG-homopolymer-tailing of the first-strand cDNA with terminaltransferase. Buckwheat FeGolS cDNAs were isolated by 5′ and 3′ RACE-PCRassays which were typically performed in either 25 or 50 μl reactionvolume containing 100 pmol primers, 200 μM dNTPs, diluted G-tailed firststrand cDNA (2 to 20 ng), 2 mM MgCl₂ in 1×PCR reaction buffer (50 mMTris/HCl, 10 mM KCl, 5 mM (NH₄)₂SO₄, pH 8.3) with 1 to 2 units ofFastStart Taq DNA Polymerase (Roche Applied Science, Indianapolis,Ind.). In the PCR assays, after the initial 4 minute denaturation stepat 94° C., 38 to 40 cycles of amplification were carried out with eachcycle consisting of the three consecutive incubations at 94° C. for 45seconds, at 50 to 58° C. for 45 seconds, and at 72° C. for 45 seconds.Finally, the assays were terminated after a 10 minute final extensioncycle at 72° C. All PCR products were cloned into pCRII-TOPO vector(Invitrogen, Carlsbad, Calif.) and propagated in Escherichia coli. Forthe isolation of cDNAs corresponding to buckwheat GolS genes, theinitial amplification was carried out using the G-tailed cDNApreparation in combination with GS1 primer(5′-GGGCCACTGAACCTTATGGGGGCACTGCTGGC-3′) (SEQ ID NO:17) representing aninternal protein coding sequence highly conserved in most GolS genes,and primer B (5′-AAGGAATTCCCCCCCCCCCCCC-3′) (SEQ ID NO:18) partiallycomplementary to the G-tailed 5′-end of the first strand cDNAs (FIG. 5).One of the amplified cDNA fragments, 469 bp in length, was shown torepresent a GolS homolog in buckwheat when its nucleotide sequence wasanalyzed by BLASTN and BLASTX programs. The gene represented by thispartial cDNA clone was designated as FeGolS-1 for Fagopyrum esculentumGolS-1. The overlapping cDNA fragments containing the 5′-end region ofFeGolS-1 cDNA were further amplified in 5′ RACE-PCR assays using anupstream internal primer, GS2 (5′-GCTCCATGATGGCTCACAGAAACAGTCC-3′) (SEQID NO:19) and primer B (FIG. 5). This PCR amplification yielded a cDNAfragment of 548 bp in length which contained the complete 5′-end of theprotein coding sequence and 82 bp long 5′ untranslated region (5′UTR).An overlapping cDNA fragment of about 900 bp in length containing thecomplete 3′-end region of FeGolS-1 was also obtained in 3′ RACE-PCRassays, using an internal primer, GS3(5′-GCTCACGCATACTATGTCATCAACTACTCC-3′) (SEQ ID NO:20) and primer A (FIG.5). In addition, two additional cDNA fragments of about 960 bp in lengthexhibiting nucleotide sequences that were nearly identical to each otherbut clearly distinct from the 3′-end region of FeGolS-1 cDNA wereobtained. Analyses of their nucleotide sequences by BLASTN and BLASTXprograms also identified them as GolS homologues. Thus, the genescorresponding to these two additional cDNAs were designated as FeGolS-2and FeGolS-3. In an attempt to amplify the 5′-end regions of theFeGolS-2 and FeGolS-3 cDNAs, 5′ RACE-PCR assays were performed usingprimer A and an internal primer, GS4(5′-GAACTTCTTGCCCTCGACCATCTTAGGCTGAG-3′) (SEQ ID NO:21) representing thenucleotide sequence that was common to FeGolS-2 and FeGolS-3 cDNA's butnot shared by FeGolS-1 cDNA (FIG. 5). An overlapping cDNA fragment of984 bp in length was obtained from the assays (FIG. 5). The nucleotidesequence of the cDNA fragment confirmed that it was a part of FeGolS-2cDNA. Finally, an intact FeGolS-1 cDNA containing the complete proteincoding sequence as well as 5′ and 3′ UTRs was reconstituted by joiningthe 398 bp long 5′-end region of the 5′ RACE-PCR clone with the 871 bplong 3′-end region of the 3′ RACE-PCR clone at the unique HindIII site(FIG. 5). Similarly, an intact FeGolS-2 cDNA was reconstituted byjoining the 700-bp 5′-end region of the 5′ RACE-PCR clone with the650-bp long 3′-end region of the 3′ RACE-PCR clone at the unique XhoIsite (FIG. 5).

DNA Sequencing

All PCR-generated cDNA clones were sequenced at the DNA SequencingFacility, BioResource Center, Cornell University(http://brcweb.biotech.cornell.edu).

Bacterial Expression and Purification of Recombinant GolS Proteins

The entire 1002 bp long protein coding sequence of FeGolS-1 cDNA wasamplified from the reconstituted FeGolS-1 cDNA using two oligonucleotideprimers, FG1-5 (5′-GTTCCAACCATATGGCACCAGAACTC-3′) (SEQ ID NO:22) andFG1-3 (5′-GGATCCGATACTTAAGCTGCGGAAGGAGC-3′) (SEQ ID NO:23) (FIG. 5).FG1-5 and FG1-3 primers contained the restriction enzyme recognitionsites for NdeI and BamHI, respectively, to allow easy cloning of theamplified coding sequence into a bacterial expression vector, pET-14b(Novagen, Madison, Wis.), in frame with the preceding poly-histidinecodons in the vector. After initial cloning into pCRIITOPO vector andamplification of the plasmid in E. coli, the protein coding sequence wasexcised from the plasmid by digestion with NdeI and BamHI, and clonedinto pET-14b vector at the corresponding cloning sites. Similarly, the1065 bp long entire protein coding sequence from the reconstitutedFeGolS-2 cDNA was inserted into pET14b vector after amplifying it withFG2-5 (5′-CATATGACTTCCGAGATGGCGCCACAG-3′) (SEQ ID NO:24) and FG2-3(5′-GGATCCTCAGGCAGCAGACGGGGCGTGTACG-3′) (SEQ ID NO:25) primers whichalso contained NdeI and BamHI sites, respectively (FIG. 5). In addition,the 987 bp long entire coding sequence was isolated from a soybean ESTclone (GenBank accession no. BE330777) presumed to encode soybeangalactinol synthase (GmGolS) in leaf tissues (INCYTE GENOMICS, cat. no.Gm-c1041), and it was cloned into pET14-b vector. Since only partialcDNA sequence data were available in GenBank, the whole cDNA insert wasre-sequenced (GenBank Accession No. AY126715). Two primers, GG-5(5′-CATCACTGAGCATATGGCTGG-3′) (SEQ ID NO:26) and GG-3(5′-GGATCCAAAGACACTCTTAAGCAGCAGATGGGG-3′) (SEQ ID NO:27), containingNdeI and BamHI restriction enzyme recognition sites, respectively, wereused for the amplification of the protein coding sequence. After cloninginto pCRIITOPO vector and amplification in E. coli, the NdeI/BamHIfragment containing the entire protein coding sequence was isolated andcloned into pET-14b vector. The pET14b plasmids containing the buckwheatand soybean GolS cDNAs were mobilized into E. coli strain BL21 (DE3)(Novagen, Madison, Wis.). Expression of the recombinant GolS proteins inE. coli were induced with 1 mM isopropyl β-D-thiogalactoside (IPTG)according to the manufacture's recommended protocol (Novagen, Madison,Wis.). The bacterial cells were collected by centrifugation, andresuspended in 10 mM Tris-HCl buffer (pH 8.0). The soluble proteinfraction was extracted from the bacterial cells by the gentle disruptionof their cell walls with BugBuster Protein Extraction Reagent (Novagen,Madison, Wis.) containing Benzonase (Novagen, Madison, Wis.). In someexperiments, the soluble protein fraction was extracted from bacterialcells through disruption of bacterial cells by sonic oscillation (at 50%level, twice for 10 seconds each, at 4° C.) with a sonicator (FisherScientific Sonic Dismembrator Model 500). Poly-histidine taggedrecombinant proteins were purified from the extracts using His.BindQuick 300 Cartridges (Novagen, Madison, Wis.) according to themanufacture's recommended protocol. Purified recombinant proteins weredialyzed against 50 mM Hepes buffer, pH 7.0, containing 5 mM MnCl₂,immediately after elution from the His.Bind Quick 300 Cartridges andbefore enzyme assay. Aliquots (0.25 to 0.5 μs) of samples of thepurified proteins were checked by SDS-PAGE using a 12% resolving gel anda 5% stacking gel. Protein samples (10 μg each) extracted from uninducedand induced bacterial cells prior to protein purification were alsoincluded in the SDS-PAGE analysis. Proteins in the gels were visualizedby staining with Coomassie Brilliant Blue R250 solution (25 g/liter inmethanol:acetic acid:water, 45:10:45, v/v/v) and destained inmethanol:acetic acid:water (30:10:60, v/v/v).

Enzyme Assays

Both the crude soluble protein extracts from E. coli and the purifiedGolS recombinant proteins were used in enzyme assays. Fagopyritolsynthase assays included 20 mM UDP-Gal as the galactosyl donor, 20 mMD-chiro-inositol as the galactosyl acceptor, 50 mM Hepes buffer, pH 7.0,2 mM dithiothreitol, 5 mM MnCl₂, and 1 to 5 μg of crude protein extractor purified enzyme protein (estimated by the Bio-Rad Protein Assay,BIO-RAD) in 50 μL total volume. In galactinol synthase assays, UDP-Galwas substituted with 20 mM galactinol as the galactosyl donor. Assayswere run at 30° C. for 30 to 300 minutes. Reactions were stopped byaddition of 50 μL of 100% ethanol. After addition of 25 μg of phenylα-D-glucoside as internal standard, the reaction mixture was heated at80° C. for 30 minutes, passed through a 10,000 MW cutoff filter(NANOSEP™ Microconcentrators, Pall Filtron Co.), and evaporated todryness under a stream of nitrogen gas. Residues were stored overnightin a desiccator with phosphorus pentoxide to remove traces of water,derivatized with trimethylsilylimidazole:pyridine (1:1, v/v) at 80° C.for 45 minutes, and analyzed for fagopyritols or other solublecarbohydrate products by high resolution gas chromatography on a HP1-MS(Agilent Technologies) capillary column (15 m length, 0.25 mm i.d., 0.25μm film thickness) as previously described (Horbowicz et al., Seed Sci.Res. 4:385-405 (1994); Horbowicz et al., Planta 205:1-11 (1994), whichare hereby incorporated by reference in their entirety).

Results

Cloning of cDNAs Encoding Two Distinct Types of GolS Enzymes inBuckwheat Seeds

Initially, several GolS gene sequences reported from various plantspecies, either derived from genomic or cDNA clones, were compiled andcompared to identify stretches of highly conserved nucleotide sequencescorresponding to the conserved amino acid domains of GolS enzymes. Byusing oligonucleotide primers representing these conserved nucleotidesequences and the first-strand cDNA synthesized from polyA⁺ RNAextracted from developing seeds in our PCR assays, a total of threedifferent GolS cDNAs from buckwheat were isolated (FIG. 5). The genescorresponding to these three buckwheat cDNA clones were designated asFeGolS-1, -2, and -3 for Fagopyrum esculentum GolS-1, -2 and -3.

FeGolS-1 cDNA was initially obtained as a partial clone of 469 bp inlength, using an internal GolS gene-specific primer (GS1) and primer Bcorresponding to the dG homopolymer tail present at the 5′ end of thecDNA (FIG. 5). Subsequently, the missing 5′-end region of FeGolS-1 cDNAwas obtained by 5′ RACE-PCR using the second internal primer (GS2) andprimer B (FIG. 5). One of the 5′ RACE-PCR clones contained a complete5′-end of the protein coding region together with 82 bp long 5′untranslated region (5′UTR) (FIG. 5). The missing 3′-end region ofFeGolS-1 cDNA was obtained by 3′ RACE-PCR using an internal primer (GS3)and primer A complementary to the polyA tail present in all cDNAs (FIG.5). In the 3′ RACE-PCR assays, two additional clones (FeGolS-2 andFeGolS-3) were obtained. They were longer (987 bp and 986 bp forFeGolS-2 and FeGolS-3, respectively) than the FeGolS-1 cDNA clone (901bp) and exhibited restriction patterns clearly distinct from that ofFeGolS-1 No obvious polyadenylation signals were found upstream of thepolyadenylation sites in any of the three genes. The 5′-end region ofFeGolS-2 cDNA containing the complete 5′-end of the protein codingregion was obtained by 5′ RACE-PCR using a gene-specific primer, GS4 andprimer B (FIG. 5). Cloning of cDNA fragments containing the 5′-end ofthe FeGolS-3 gene was not successful.

Intact FeGolS-1 and FeGolS-2 cDNAs containing the complete proteincoding sequences with 5′ and 3′ UTRs were reconstituted by joining theoverlapping 5′ and 3′ RACE-PCR clones for each gene (FIG. 5). Thereconstituted FeGolS-1 cDNA is 1269 bp long containing a single openreading frame (ORF) (GenBank accession no. AY126718). On the other hand,the reconstituted FeGolS-2 cDNA is 1326 bp long; it also contains asingle ORF (GenBank Accession No. AY126716). The partial FeGolS-3 cDNAclone is 986 bp long and contains the complete 3′-end of the cDNA(GenBank accession no. AY126717). According to the nucleotide sequencecomparison, FeGolS-1 is distinct from FeGolS-2 sharing only 62.2%sequence identity. On the other hand, FeGolS-2 and FeGolS-3 share anearly identical nucleotide sequence in their 3′ regions. Whereas theFeGolS-2 cDNA clone differs from FeGolS-3 only by 15 nucleotides withinthe 986/987 bp long 3′ region, FeGolS-2 differs from FeGolS-1 by 385nucleotides at the corresponding 3′ region. These results suggest thatFeGolS-1 and FeGolS-2 represent two different members of a GolS genefamily in buckwheat. The complete 1406 bp nucleotide sequence of thesoybean galactinol synthase (GmGolS) cDNA (assigned GenBank AccessionNo. AY126715) had a high degree of sequence similarity to FeGolS-1.

Primary Structures of GolS Polypeptides Deduced from cDNA Sequences

The amino acid sequence deduced from the reconstituted FeGolS-1 cDNAindicated that it is capable of encoding a polypeptide of 333 amino acidresidues with a predicted molecular mass of 38.3 kDa (FIG. 1). On theother hand, FeGolS-2 cDNA is capable of encoding a polypeptide of 354amino acids with a predicted molecular mass of 40.7 kDa (FIG. 2).Predicted FeGolS-2 and -3 differ from each other only by three aminoacid residues in the carboxyl half of the polypeptide whereas eachdiffers from FeGolS-1 by 96 amino acid residues in the correspondingregion. The presence of a longer stretch (additional 17 residues) ofamino acid sequence was identified near the carboxyl termini in FeGolS-2(and also in FeGolS-3), mainly accounting for its larger predictedmolecular mass than that for FeGolS-1 (FIG. 6). The amino acid sequencededuced from the 987 bp long coding sequence of the soybean GmGolS cDNAindicated that it is capable of encoding a polypeptide of 328 amino acidresidues with a predicted molecular mass of 38.0 kDa (FIG. 4).

Both FeGolS-1 and FeGolS-2 polypeptides share a high degree of aminoacid sequence similarity with other GolSs identified from a wide varietyof plant species (FIG. 7). The highly conserved serine phosphorylationsite and the carboxyl terminal pentapeptide, APSAA (SEQ ID NO: 28)(Sprenger et al., Plant 21:249-258 (2000), which is hereby incorporatedby reference in its entirety) are also present in all three FeGolSproteins. In addition, a putative manganese binding motif, DXD, believedto be conserved in most galactosyl transferases (Breton et al., J.Biochem. 123:1000-1009 (1998); Busch et al., J. Biol. Chem.273:19566-19572 (1998); Wiggins et al., Proc. Natl. Acad. Sci. USA95:7945-7950 (1998), which are hereby incorporated by reference in theirentirety) is also present in all GolSs examined, including the threeFeGolSs. A phylogenetic analysis indicated that both FeGolS-1 andFeGolS-2 are evolutionarily most closely related to a Brassica napusGolS.

Recombinant Protein Expression and Purification

FIG. 8 shows an SDS-PAGE gel used to monitor the protein expression andpurification steps. Total soluble protein extracts from uninduced andinduced bacteria cells harboring FeGolS-1 cDNA are shown in lanes 2 and3, respectively. The purified recombinant FeGolS-1 protein fraction(lane 4) contained a single prominent polypeptide with an apparentmolecular mass of 43 kDa. Total soluble protein extracts from uninducedand induced bacteria cells harboring FeGolS-2 cDNA are shown in lanes 5and 6, respectively. The purified recombinant FeGolS-2 protein fraction(lane 7) contained a single prominent polypeptide with an apparentmolecular mass of 45.5 kDa. Total soluble protein extracts fromuninduced and induced bacteria cells harboring GmGolS cDNA are shown inlanes 8 and 9, respectively. A single polypeptide of with an apparentmolecular mass of 43 kDa was found in the purified recombinant GmGolSprotein fraction (lane 10). No polypeptide with its molecular masscorresponding to any of the recombinant GolS proteins described abovewas found after purification of histidine-tagged protein from the totalsoluble protein extract from control bacteria which had been transformedwith the pET-14b vector alone. These results indicated that the purifiedrecombinant FeGolS-1, FeGolS-2, and GmGolS proteins were derived fromthe expression of their corresponding genes.

Substrate Specificity of FeGolS-1 and FeGolS-2

Both purified recombinant FeGolS-1 and FeGolS-2 proteins exhibitedfagopyritol synthase activities. FeGolS-1 catalyzed the biosynthesis offagopyritol B1 with UDP-Gal as the galactosyl donor and D-chiro-inositolas the galactosyl receptor (FIG. 9A). However, only FeGolS-2 catalyzedthe biosynthesis of both fagopyritol A1 and fagopyritol B1 in a ratio of1:4 demonstrating the unique product specificity of FeGolS-2 (FIG. 9B).Both FeGolS-1 and FeGolS-2 catalyzed the biosynthesis of galactinol withUDP-Gal as galactosyl donor and myo-inositol as galactosyl receptor(FIGS. 9D and 9E), consistent with the structural homology of theseenzymes to galactinol synthase. No products were biosynthesized usingprotein extracts from control bacteria transformed with the vector only,confirming that FeGolS-1 and FeGolS-2 catalyzed the biosynthesis offagopyritols and galactinol. Neither FeGolS-1 nor FeGolS-2 was activewith galactinol as the galactosyl donor, demonstrating that both enzymeshad substrate specificity for UDP-Gal. Neither FeGolS-1 nor FeGolS-2biosynthesized fagopyritol A1 from fagopyritol B1 (as both donor andreceptor) indicating that FeGolS-2 catalyzes the biosynthesis offagopyritol A1 directly by transfer of the galactosyl residue fromUDP-Gal. As a control, soybean galactinol synthase (GmGolS) catalyzedthe biosynthesis of galactinol with UDP-Gal and myo-inositol assubstrates (FIG. 9F), but also catalyzed the biosynthesis of fagopyritolB1, but not fagopyritol A1, with UDP-Gal and D-chiro-inositol assubstrates (FIG. 9C). Activity of FeGolS-1 was similar to that forGmGolS, whereas FeGolS-2, by catalyzing the biosynthesis of fagopyritolA1, was uniquely different from the soybean enzyme.

Discussion

The FeGolS-1 gene encodes an enzyme that catalyzes fagopyritol B1biosynthesis using UDP-Gal as galactosyl donor and D-chiro-inositol asgalactosyl acceptor. The FeGolS-2 gene, a unique member of the buckwheatgalactinol synthase gene family, encodes a fagopyritol synthase thatcatalyzes the biosynthesis of both fagopyritol A1 and fagopyritol B1using UDP-Gal as galactosyl donor and D-chiro-inositol as galactosylacceptor. Based on the molecular structure and absolute configuration offagopyritol A1 and fagopyritol B1 determined by NMR (Obendorf et al.,Carbohydr. Res. 328:623-627 (2000), which is hereby incorporated byreference in its entirety), FeGolS-2 catalyzes the formation of theα-(1→3)-linkage unique to fagopyritol A1 and other members of thefagopyritol A series found only in buckwheat, as well as theα-(1→2)-linkage of fagopyritol B1 and other members of the fagopyritol Bseries (Obendorf et al., Carbohydr. Res. 328:623-627 (2000); Steadman etal., Carbohydr. Res. 331:19-25 (2001), which are hereby incorporated byreference in their entirety). FeGolS-1, FeGolS-2, and GmGolS allbiosynthesize galactinol using UDP-Gal as galactosyl donor andmyo-inositol as galactosyl acceptor. However, buckwheat FeGolS-1 andsoybean GmGolS do not form fagopyritol A1. Thus, the novel buckwheatFeGolS-2 gene and its protein product are distinctly different in bothstructure and function from the buckwheat FeGolS-1 gene and the soybeanGmGolS gene and their corresponding proteins. The longer amino acidsequence (13 to 23 amino acids) near the carboxyl end of buckwheatFeGolS-2 (and also FeGolS-3) is unique among known GolS sequences fromvarious species and may be related to the property of FeGolS-2 to formthe unique α-(1→3)-linkage.

Retention of Fagopyritol Synthase Activity by Purified Recombinant

FeGolS-1, FeGolS-2, and GmGolS protein required Mn⁺² (5 mM optimal) as acofactor, as it has been reported with galactinol synthase from othersources (Saravitz et al., Plant Physiol. 83:185-189 (1987); Castillo etal., J. Agric. Food Chem. 38:351-355 (1990); Smith et al., PlantPhysiol. 96:693-698 (1991); Liu et al., Plant Physiol. 109:505-511(1995); Kuo et al., Plant Sci. 125:1-11 (1997), which are herebyincorporated by reference in their entirety). One to 10 mM Mn⁺² was mostcommonly used for the retention of galactinol synthase activity.Interestingly, the antihyperglycemic effects of D-chiro-inositol wereassociated with manganese (Fonteles et al., Hormone Metab. Res.32:129-132 (2000), which is hereby incorporated by reference in itsentirety) in subjects with non-insulin dependent diabetes melitus.Buckwheat seeds are a rich source of manganese (Steadman et al., J. Sci.Food Agric. 81:1094-1100 (2001), which is hereby incorporated byreference in its entirety), and buckwheat has been used for thetreatment of diabetes (Lu et al., in Proceedings of the 5thInternational Symposium on Buckwheat, eds. Lin et al., AgriculturePublishing House, Beijing, pp 458-464 (1992); Wang et al., inProceedings of the 5th International Symposium on Buckwheat, eds. Lin etal., Agriculture Publishing House, Beijing, pp 465-467 (1992), which arehereby incorporated by reference in their entirety).

Pea (Pisum sativum L.) seed galactinol synthase (Frydman et al.,Biochem. Biophys. Res. Comm. 12:121-125 (1963), which is herebyincorporated by reference in its entirety) and lentil (Lens culinarisMedik.) stachyose synthase (Hoch et al., Arch. Biochem. Biophys.366:75-81 (1999), which is hereby incorporated by reference in itsentirety) have been reported to form a product with D-chiro-inositol assubstrate, but the product was not confirmed to be a fagopyritol. Thelack of activity of Adzuki bean (Vigna angularis Ohwi and Ohashi)stachyose synthase with D-chiro-inositol (Peterbauer et al., PlantPhysiol. 117:165-172 (1998), which is hereby incorporated by referencein its entirety) and the very limited accumulation of stachyose inbuckwheat seeds (Horbowicz et al., Planta 205:1-11 (1998), which ishereby incorporated by reference in its entirety) suggest that stachyosesynthase is not involved in the biosynthesis of fagopyritols. Theresults reported herein clearly demonstrate that FeGolS-2, a galactinolsynthase homologue, catalyzes the biosynthesis of both fagopyritol A1and fagopyritol B1.

Among seven GolS genes identified in Arabidopsis thaliana, three wereidentified as stress responsive (Taji et al., Plant J. 29:417-426(2002), which is hereby incorporated by reference in its entirety).AtGolS-1 and AtGolS-2 were induced by drought and high-salinity stressesbut not by cold stress. In contrast, AtGolS-3 was induced by cold stressby not by drought or high-salinity stress. Buckwheat seeds matured at18° C. accumulated more fagopyritol A1 and fagopyritol B1 than seedsmatured at 25° C. (Horbowicz et al., Planta 205:1-11 (1998), which ishereby incorporated by reference in its entirety), indicating thatFeGolS genes may be cold-responsive.

The nucleotide sequence of the soybean EST clone, BE330777, isolated bya public source (Shoemaker et al., Public soybean EST project; GenBankBE33077; Genome Systems Clone ID: Gm-c1041-80 (5′), Genome Systems,Inc., 4633 World Parkway Circle, St. Louis, Mo. 63134 (1999), which ishereby incorporated by reference in its entirety) with the full sequencefirst reported herein, demonstrated a very high homology to the soybeanseed galactinol synthase gene (155634), sequence 6 (U.S. Pat. No.5,648,210 to Kerr et al., which is hereby incorporated by reference inits entirety). The deduced amino acid sequence (328 amino acids)differed by only one amino acid, Ile 223 in GmGolS (AY126715) ratherthan Met 223 (155634) (U.S. Pat. No. 5,648,210 to Kerr et al., which ishereby incorporated by reference in its entirety). Of the multiple genesfor galactinol synthase, some are specifically expressed in seeds.Modification of galactinol biosynthesis is of commercial interest (U.S.Pat. No. 5,648,210 to Kerr et al.; U.S. Pat. No. 5,710,365 to Kerr,which are hereby incorporated by reference in their entirety) forproducing soybean seeds with lower stachyose concentrations for thepoultry and pig feed industry (Sebastian et al., in Soy in AnimalNutrition, ed. Drackley, Federation of Animal Science Societies, Savoy,Ill., pp 56-73 (2000), which is hereby incorporated by reference in itsentirety). A mutant with a single base change in a seed-expressedmyo-inositol 1-phosphate synthase (MIPS, EC 5.5.1.4) gene coupled withappropriate modifiers resulted in soybean seeds with both reduced phyticacid and reduced stachyose (Hitz et al., Plant Physiol. 128:650-660(2002), which is hereby incorporated by reference in its entirety) foruse in the feed industry.

Fagopyritol A1 is isosteric with2-amino-2-deoxy-α-D-galactopyranosyl-(1→3)-D-chiro-inositol (Berlin etal., Tetrahedron Lett. 31:1109-1112 (1990), which is hereby incorporatedby reference in its entirety) related to a putative insulin mediator(Lamer et al., Biochem. Biophys. Res. Comm. 151:1416-1426 (1988), whichis hereby incorporated by reference in its entirety) deficient insubjects with NIDDM and PCOS. The novel FeGolS-2 gene and FeGolS-2enzyme described herein may be used to form the unique α-(1→3)-linkagebetween galactose and D-chiro-inositol.

Example 2 Seed Galactosyl Cyclitols Enhanced by Substrate Feeding

Materials and Methods

Plant Materials

Soybean (Glycine max (L.) Merrill) plants were grown in the greenhouse(Obendorf et al., Crop Sci. 20:483-486 (1980); Obendorf et al., CropSci. 38:78-84 (1998), which are hereby incorporated by reference intheir entirety) at 27° C. during the day (14 hours) and 22° C. at night(10 hours) under natural sunlight supplemented 14 hours daily with 640μmol m⁻² s⁻¹ incandescent light from metal halide lamps (Sylvania 1000watt BU). Three embryos isolated from immature seeds (250±20 mg freshweight, approximately 35 DPA) by removal of the seed coat and nucellusremnants were incubated in 20 mL screw-capped vials containing 3 mL ofsubstrate (cyclitol and/or sucrose) solutions for 24 hours at 25° C. and200 μmol m⁻² s⁻¹ fluorescent light. Embryos were blotted, placed insmall plastic Petri dishes, and subjected to slow drying at 22° C. bydaily transfer to successive lower relative humidity (RH) controlled bysaturated salt solutions (Blackman et al., Plant Physiol. 100:225-230(1992), which is hereby incorporated by reference in its entirety): day1, 92% RH; day 2, 87% RH; day 3, 75% RH; day 4, 54% RH; day 5, 45% RH;day 6, 32% RH; day 7, 12% RH; and remained at 12% RH days 8-14.

Embryo Feeding Experiments—Substrate Concentration Series

Four substrate concentration experiments were conducted. Embryos foreach experiment were incubated in each of the substrate solutions for 24hours, blotted, and slow dried for 14 days. Axis and cotyledon tissueswere separated and analyzed for soluble carbohydrates. Four replicationsof three embryos each (total of 12 embryos/treatment) were incubated inthe myo-inositol-sucrose concentration series: A) 0 mM myo-inositol+100mM sucrose, B) 10 mM myo-inositol+90 mM sucrose, C) 25 mMmyo-inositol+75 mM sucrose, D) 50 mM myo-inositol+50 mM sucrose, E) 100mM myo-inositol+0 mM sucrose, and F) 0 mM myo-inositol+0 mM sucrose. Sixreplications of three embryos each (total of 18 embryos/treatment) wereincubated in the D-chiro-inositol-sucrose concentration series, andthree replications of three embryos each (total of 9 embryos/treatment)were incubated in the D-pinitol-sucrose concentration series. TreatmentsA) through F) were identical in both concentration series, except forthe substitution of D-chiro-inositol or D-pinitol instead ofmyo-inositol. In the sucrose concentration series, three replications ofthree embryos (total of 9 embryos/treatment) were incubated with 0, 25,50, 75, 100, and 200 mM sucrose.

Embryo Feeding Experiments—Drying Time Series

Six slow drying time experiments were conducted. In each experiment,three replications of three embryos each (total of 9 embryos pertreatment) were incubated in a different sucrose and/or cyclitolsubstrate solution for 24 hours, blotted, and slow dried for 0, 1, 2, 3,4, or 14 days. Axis and cotyledon tissues were separated and analyzedfor soluble carbohydrates. The substrate solutions for the sixexperiments were as follows: 30 mM myo-inositol plus 100 mM sucrose; 100mM D-chiro-inositol; 100 mM D-pinitol; 100 mM D-pinitol plus 100 mMD-chiro-inositol; 50 mM D-pinitol plus 50 mM D-chiro-inositol; and 100mM D-pinitol plus 100 mM D-chiro-inositol plus 100 mM sucrose.

Substrates

Sucrose, myo-inositol, scyllo-inositol, epi-inositol, and UDP-Gal werepurchased from Sigma-Aldrich (St. Louis, Mo.). D-Pinitol,D-chiro-inositol, L-chiro-inositol, D-ononitol, and L-quebrachitol werepurchased from Industrial Research Limited (Lower Hutt, New Zealand).Sequoyitol was purchased from Carl Roth GmbH & Co. KG (Karlsruhe,Germany). Bornesitol was purified from seeds of Lathyrus odoratus L.Galactinol was purified from lemon balm (Melissa officinalis L.) leaves.When needed, substrates were purified by carbon-Celite columnchromatography (Whistler et al., J. Amer. Chem. Soc. 72:677-679 (1950),which is hereby incorporated by reference in its entirety) before use.Carbon was purchased from Mallinckrodt Baker Inc (Phillipsburg, N.J.).Celite was purchased from Supelco (Bellefonte, Pa.).

Carbohydrate Analysis

Soluble carbohydrates were extracted from 2 cotyledons or 1 axis foreach embryo. Two cotyledons were extracted with 2.0 mL of ethanol:water(1:1, v/v) containing 300 μg of phenyl α-D-glucoside as internalstandard. One axis was extracted with 1.0 mL of ethanol:water (1:1, v/v)containing 100 μg of phenyl α-D-glucoside as internal standard. Extractswere passed through a 10,000 molecular weight cut-off filter (NANOSEP10K Omega, Paul Filton Co., Northborough, Mass.) by centrifugation, and200 μL were dried in silylation vials under nitrogen gas, derivatizedwith 200 μL of trimethylsilylsylimidazole:pyridine (1:1, v/v), andanalyzed by high resolution gas chromatography on a HP1-MS (AgilentTechnologies, Palo Alto, Calif.) capillary column (15 m length, 0.25 mmi.d., 0.25 μm film thickness) as previously described (Horbowicz et al.,Seed Sci. Res. 4:385-405 (1994), which is hereby incorporated byreference in its entirety).

Results

Cyclitols, including myo-inositol, D-chiro-inositol, and D-pinitol, werefed to immature soybean embryos followed by precocious maturationinduced by slow-drying of the embryos and analysis of solublecarbohydrates in axis and cotyledon tissues. Exogenously fed freecyclitols were taken up by embryo tissues. In 250 mg fresh weightembryos, initial concentrations of cyclitols in axis and cotyledontissues, respectively, were myo-inositol 10.9 and 11.0 mg/g dry weight,D-chiro-inositol 1.4 and 1.2 mg/g dry weight, and D-pinitol 6.0 and 4.0mg/g dry weight. After incubation with 30 mM myo-inositol, 100 mMD-chiro-inositol, or 100 mM D-pinitol for 24 hours at 22° C.,concentrations of myo-inositol increased 1.8 fold in axis and 2 fold incotyledon tissues, D-chiro-inositol increased 18 fold and 40 fold, andD-pinitol increased 6 fold and 11 fold, respectively.

Both embryonic axis and cotyledon tissues were assayed for experimentsreported herein. Embryonic axes mature earlier than cotyledons andaccumulate higher concentrations of soluble carbohydrates (up to 25% ofdry weight) (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Obendorfet al., Crop Sci. 38:78-84 (1998), which are hereby incorporated byreference in their entirety). Accumulation of products in axis tissuesgenerally precedes accumulation of products in cotyledons, reflectingthe differential in progression toward maturation. In general, data weremore variable for axis tissues than for cotyledon tissues, mainlybecause of the small mass of axis tissues, about 1 mg dry weight forexperiments reported herein.

Concentration series experiments were adjusted to be a constant 100 mM(cyclitol plus sucrose) excluding the sucrose concentration seriesexperiment. Feeding myo-inositol up to 50 mM doubled free myo-inositolconcentration in dry axis and cotyledon tissues after precociousmaturation with small increases in D-pinitol and D-chiro-inositol (FIGS.10A and D). Galactinol accumulation doubled in cotyledons after feeding25 to 50 mM myo-inositol while fagopyritol B1 accumulation was reduced(FIG. 10E), demonstrating a competition between the biosynthesis ofgalactinol and fagopyritol B1. There was little change in galactopinitolA, galactopinitol B, raffinose, or stachyose concentrations in eitheraxis or cotyledon tissues after feeding myo-inositol (FIGS. 10B, C, E,and F). In the absence of exogenous sucrose, sucrose concentration inaxis tissues was reduced to 50%, but sucrose concentration in cotyledonsremained constant (FIGS. 10C and F). These results are consistent withthe role of myo-inositol as a substrate in galactinol biosynthesis and aproduct in the biosynthesis of raffinose and stachyose in seeds (FIG.11). Feeding 30 mM myo-inositol and 100 mM sucrose together resulted inelevated amounts of free myo-inositol during day 1 of slow drying andthen a decline in myo-inositol (FIGS. 12A and D), a transient increasein galactinol during days 2 and 3 (FIGS. 12B and E), and then a declinein galactinol as raffinose and stachyose accumulated (FIGS. 12C and F).The decrease in total myo-inositol indicates metabolism of myo-inositolto other products, including phytin and cell walls, within the embryo(Loewus et al., Plant Sci. 150:1-19 (2000); Hegeman et al., PlantPhysiol. 125:1941-1948 (2001); Hitz et al., Plant Physiol. 128:650-660(2002), which are hereby incorporated by reference in their entirety).

Feeding D-chiro-inositol resulted in a 40- to 50-fold increase in freeD-chiro-inositol concentration in axis and cotyledons (FIGS. 13A and D),a 17-fold increase in fagopyritol B1 concentration in axis tissues and a7-fold increase in cotyledons (FIGS. 13B and E), but did not increaseD-pinitol, myo-inositol, galactopinitol A, galactopinitol B, galactinol,raffinose, or stachyose concentrations (FIG. 13). The highconcentrations of free D-chiro-inositol declined (FIGS. 14A and D) and alarge increase in fagopyritol B1 occurred between day 2 and day 4 ofslow drying accompanied by the decrease in concentration of freeD-chiro-inositol in axis and cotyledon tissues (FIGS. 14A, B, D, and E).A transient accumulation of galactinol signaled an accumulation ofraffinose and stachyose and modest accumulation of galactopinitol A andgalactopinitol B (FIGS. 14B, C, E, and F, compared to FIG. 12). Theseresults suggest that D-chiro-inositol does not serve as precursor tomyo-inositol or D-pinitol in soybean embryos, and that fagopyritol B1does not serve as an alternate galactosyl donor for the biosynthesis ofraffinose and stachyose. The large increase in fagopyritol B1 fromexternally applied D-chiro-inositol suggests that D-chiro-inositol isnot biosynthesized within the embryo but is transported to the embryofrom maternal tissues. The increase in sucrose during slow drying (FIGS.14C and F) probably reflects starch degradation within the embryo.

Feeding D-pinitol resulted in an 8-fold increase in free D-pinitolconcentration (FIGS. 15A and D) and a more than 4-fold increase in bothgalactopinitol A and galactopinitol B concentrations (FIGS. 15B and E).Concentrations of D-chiro-inositol, myo-inositol, fagopyritol B1,galactinol, raffinose, and stachyose were not increased (FIG. 15).Feeding 100 mM D-pinitol resulted in high concentrations of freeD-pinitol and a substantial increase in galactopinitol A andgalactopinitol B between day 2 and day 4 of slow drying (FIGS. 16A, B,D, and E). A transient increase in galactinol occurred as raffinose andstachyose accumulated (FIGS. 16B, C, E, and F). The larger increase instachyose in cotyledons, compared to feeding D-chiro-inositol (FIG.14F), suggests that galactopinitol A may be effective as a galactosyldonor for stachyose biosynthesis as suggested by Hoch et al., Arch.Biochem. Biophys. 366:75-81 (1999) and Peterbauer et al., Seed Sci. Res.11:185-198 (2001), which are hereby incorporated by reference in theirentirety. The large increase in galactopinitols from externally appliedD-pinitol suggests that D-pinitol is not biosynthesized within theembryo but is transported to the embryo from maternal tissues. Sucroseconcentration increased through day 3 of slow drying (FIGS. 16C and F).

Feeding sucrose at 0 to 200 mM resulted in a small decrease ingalactinol (FIG. 17B) and an increase in sucrose in axis tissues (FIG.17C) but little change in concentrations of soluble carbohydrates incotyledon tissues (FIG. 17). These results suggest that osmoticconcentrations, per se, have little effect on soluble carbohydrateconcentrations under the experimental conditions used in theseexperiments.

Feeding a combination of 100 mM D-pinitol and 100 mM D-chiro-inositolresulted in high concentrations of both free D-pinitol and freeD-chiro-inositol; free D-chiro-inositol declined with elevatedconcentrations of fagopyritol B1, D-pinitol decreased less but withincreases in galactopinitol A, galactopinitol B, stachyose, andraffinose between day 2 and day 3 in embryo cotyledon tissues (FIG. 18).Galactinol concentration peaked by day 1 (axis) or day 2 (cotyledons)and declined as raffinose, stachyose, and galactopinitols accumulated.

Accumulation of fagopyritol B1 appeared to be independent ofaccumulation of galactopinitols, raffinose, and stachyose, indicatingfagopyritol B1 biosynthesis is independent of galactopinitolbiosynthesis. Feeding a combination of D-pinitol and D-chiro-inositol(FIG. 18) resulted in a 50% decrease (14 days in steady state galactinolconcentration and a 50% decrease (14 days) in galactopinitol A plusgalactopinitol B concentration in cotyledons (FIG. 18E), compared tofeeding D-pinitol alone (FIG. 16E), but only a 10 to 15% decrease (4days) in fagopyritol B1 concentration (FIG. 18E) compared to feedingD-chiro-inositol alone (FIG. 14E) (14 versus 16 mg/g DW at day 4 of slowdrying). In axis tissues, galactinol, galactopinitol A, galactopinitolB, raffinose, and stachyose were not decreased by feeding a combinationof D-pinitol and D-chiro-inositol (FIGS. 18C and D) compared to feedingD-pinitol alone (FIGS. 16C and D). Fagopyritol B1 in axis tissues wasreduced about 50% (14 days) after feeding a combination of D-pinitol andD-chiro-inositol (FIG. 18B) compared to feeding D-chiro-inositol alone(FIG. 14B). In all cases, fagopyritol B1 was maximum in axis tissues onday 3 of slow drying while in cotyledons fagopyritol B1 continued toincrease during day 4 of slow drying. The small mass of the axis tissues(approximately 1 mg dry weight) may have hastened the cessation ofgalactosyl cyclitol accumulation in axis tissues compared to cotyledonsduring precocious maturation. In addition, axis tissues yellowed 1 to 2days sooner during precocious maturation after feeding D-pinitol orcombinations of D-pinitol and D-chiro-inositol than after feedingD-chiro-inositol alone. Feeding a combination of 50 mM D-pinitol plus 50mM D-chiro-inositol resulted in patterns identical to those with 100 mM(FIG. 18), indicating the cyclitol substrates were at saturatingconcentrations. Feeding a combination of 100 mM D-pinitol, 100 mMD-chiro-inositol, and 100 mM sucrose resulted in patterns identical tothose without sucrose (FIG. 18), except that sucrose concentrations werehigher initially.

Cyclitols detected in soybean embryos include myo-inositol, D-pinitol,and D-chiro-inositol (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994);Obendorf et al., Plant Sci. 132:1-12 (1998); Obendorf et al., Crop Sci.38:78-84 (1998), which are hereby incorporated by reference in theirentirety). If present, other cyclitols were below the level ofdetection. myo-Inositol is biosynthesized in soybean embryos, andinhibition of myo-inositol biosynthesis results in reduced accumulationof phytic acid, galactinol, raffinose, and stachyose (Hegeman et al.,Plant Physiol. 125:1941-1948 (2001); Hitz et al., Plant Physiol.128:650-660 (2002), which are hereby incorporated by reference in theirentirety). Total D-chiro-inositol or total D-pinitol did not increase inthe absence of exogenous feeding of the corresponding cyclitols,consistent with our previous results with soybean zygotic embryosmatured in vitro (Obendorf et al., Plant Sci. 132:1-12 (1998); Obendorfet al., Crop Sci. 38:78-84 (1998), which are hereby incorporated byreference in their entirety) and indicating a lack of biosynthesis ofboth D-chiro-inositol and D-pinitol during precocious maturation ofsoybean zygotic embryos.

Axis tissues accumulate higher concentrations of soluble carbohydrateproducts than cotyledons, suggesting that biosynthetic enzymes may bemore active in axis tissues. Yellowing of axis and cotyledon tissues isa visual indicator of the cessation of growth and tissue maturation;axis tissues mature earlier than cotyledon tissues in planta (Obendorfet al., Crop Sci. 38:78-84 (1998), which is hereby incorporated byreference in its entirety). This difference in maturation must beconsidered when assaying gene expression in whole embryos or seeds incontrast to assaying axis and cotyledon tissues separately. FeedingD-pinitol or combinations of D-pinitol and D-chiro-inositol resulted inyellowing of axis tissues 1 to 2 days earlier during precociousmaturation than feeding D-chiro-inositol alone. Because of their smallsize and more rapid maturation rate after feeding D-pinitol,precociously matured axis tissues may not reflect product accumulationpatterns as accurately as cotyledons. Therefore, more emphasis should beplaced on the product accumulation patterns in precociously maturedcotyledons.

Feeding both D-pinitol and D-chiro-inositol reduced galactinolconcentration in cotyledons by 50% compared to feeding D-pinitol alone,indicating a competition between the biosynthesis of fagopyritol B1 andgalactinol by GolS. The 50% reduction in the biosynthesis ofgalactopinitols reflects the 50% reduction in galactinol, the galactosyldonor for galactopinitol biosynthesis by stachyose synthase (Peterbaueret al., Seed Sci. Res. 11:185-198 (2001), which is hereby incorporatedby reference in its entirety). The small decrease in fagopyritol B1biosynthesis after feeding both D-pinitol and D-chiro-inositol comparedto feeding D-chiro-inositol alone, probably reflects competition foravailable UDP-Gal between galactinol and fagopyritol B1 biosynthesis.Results of substrate feeding experiments are consistent with theinterpretation that D-pinitol and D-chiro-inositol are transported frommaternal tissues and not biosynthesized in the embryo tissues. Inaddition, galactopinitols and fagopyritol B1 are biosynthesized bydifferent pathways, fagopyritols are biosynthesized by GolS,galactopinitols are biosynthesized by stachyose synthase/raffinosesynthase, and galactopinitols may serve as galactosyl donors forstachyose biosynthesis.

Example 3 Soybean EST Clone Corresponding to Galactinol Synthase (GoFS)Gene

Gene or cDNA sequences corresponding to the GolS gene in soybean weresearched in the nucleotide and protein databases using the BLASTprograms (http://www.ncbi.nlm.nih.gov) and a multiple sequence alignmentprogram, CLUSTAL W (http://workbench.sdsc.edu). A soybean EST clone(GenBank accession number BE330777) sharing a very high level of DNAsequence identity with the GolS genes reported from other plant specieswas identified, and obtained from INCYTE GENOMICS, Palo Alto, Calif.(cat. no. Gm-c1041). Since only partial DNA sequence data were availablefor this EST clone in GenBank, the whole EST insert was re-sequenced(nucleotide sequence assigned to GenBank Accession Number AY126715) atthe DNA Sequencing Facility at BioResource Center at Cornell University(http://brcweb.biotech.cornell.edu).

The 987 bp long entire protein coding sequence of GmGolS was amplifiedfrom the soybean EST clone by PCR. Two primers,5′-CATCACTGAGCATATGGCTGG-3′ (SEQ ID NO:29) and5′-GGATCCAAAGACACTCTTAAGCAGCAGATGGGG-3′ (SEQ ID NO:30), containing NdeIand BamHI restriction enzyme recognition sites respectively, were usedin the PCR assays. After cloning into the pCRII-TOPO vector (Invitrogen,Carlsbad, Calif.) and amplification in Escherichia coli, the NdeI/BamHIfragment containing the entire protein coding sequence was isolated andcloned into the corresponding sites in pET-14b vector (Novagen, Madison,Wis.). This insertion resulted in the placement of the GmGolS proteincoding sequence in frame with the preceding poly-histidine codons in thepET-14b vector. The pET14b plasmid containing soybean GmGolS cDNA wasmobilized into E. coli strain BL21 (DE3) (Novagen, Madison, Wis.).Expression of the recombinant GmGolS protein was induced in E. coli with1 mM isopropylthio-β-D-galactoside (IPTG) according to themanufacturer's recommended protocol (Novagen, Madison, Wis.). Thebacterial cells were collected by centrifugation, and resuspended in 10mM Tris-HCl buffer (pH 8.0). The soluble protein fraction was extractedfrom the bacterial cells by the gentle disruption of their cell wallswith BugBuster Protein Extraction Reagent (Novagen, Madison, Wis.)containing Benzonase (Novagen, Madison, Wis.). Poly-histidine taggedrecombinant proteins were purified from the extracts using His.BindQuick 900 Cartridges (Novagen, Madison, Wis.) according to themanufacturer's recommended protocol. Purification of proteins wasverified by SDS-PAGE. Purified recombinant proteins were dialyzedagainst 50 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid]-NaOH buffer, pH 7.0, containing 5 mM MnCl₂, immediately afterelution from the His.Bind Quick 900 Cartridges and prior to enzymeassay.

Both crude soluble protein extracts from E. coli containing therecombinant GmGolS protein and purified recombinant GmGolS protein wereused in enzyme assays. GolS activity assays included 20 mM UDP-Gal asthe galactosyl donor, 20 mM myo-inositol as the galactosyl acceptor, 50mM HEPES buffer, pH 7.0, 2 mM DTT, 5 mM MnCl₂ and 1 to 5 μg of crudeprotein extract or purified GmGolS protein in 50 μL total volume. Infagopyritol synthase assays, myo-inositol was substituted with 20 mMD-chiro-inositol as the galactosyl acceptor. Assays were run at 30° C.for 30 to 300 minutes. Reactions were stopped by addition of 50 μL of100% ethanol. After addition of 25 μg of phenyl α-D-glucoside asinternal standard, the reaction mixture was heated at 80° C. for 30minutes, passed through a 10,000 MW cutoff filter (NANOSEP), andevaporated to dryness under a stream of nitrogen gas. Residues werestored overnight in a desiccator with phosphorus pentoxide to removetraces of water, derivatized with trimethylsilylimidazole:pyridine (1:1,v/v) at 80° C. for 45 minutes, and analyzed for fagopyritols or othersoluble carbohydrate products by high resolution gas chromatography on aHP1-MS (Agilent Technologies) capillary column as previously described(Horbowicz et al., Seed Sci. Res. 4:385-405 (1994), which is herebyincorporated by reference in its entirety).

To confirm that GolS catalyzes the biosynthesis of fagopyritol B1, asoybean galactinol synthase (GmGolS) gene was cloned (GenBank accessionnumber AY126715) and heterologously expressed in Escherichia coli. Thepurified recombinant protein was assayed for fagopyritol synthaseactivity. Recombinant GmGolS catalyzed the biosynthesis of galactinolwith UDP-Gal as the galactosyl donor and myo-inositol as the galactosylacceptor (FIG. 19A), but also catalyzed the biosynthesis of fagopyritolB1 with UDP-Gal as the galactosyl donor and D-chiro-inositol as thegalactosyl receptor (FIG. 19B). GmGolS was not active with galactinol asthe galactosyl donor. Using UDP-Gal as the galactosyl donor, GmGolS wasnot active with O-methylated cyclitols including D-pinitol(1D-3-O-methyl-chino-inositol), D-ononitol (1D-4-O-methyl-myo-inositol),sequoyitol (5-O-methyl-myo-inositol), or L-quebrachitol(1L-2-O-methyl-chiro-inositol) as galactosyl acceptors, except forreduced activity with D-bornesitol (1D-1-O-methyl-myo-inositol). GmGolSwas active with L-chiro-inositol as the galactosyl acceptor, but hadreduced activity with scyllo-inositol and no activity with epi-inositolusing UDP-Gal as galactosyl donor.

The recombinant soybean galactinol synthase (GmGolS) is amulti-functional enzyme with both GolS activity and fagopyritol synthaseactivity, but GmGolS does not exhibit galactopinitol synthase activity.GolS activity in developing and maturing soybean seeds is associatedwith stachyose accumulation and remained high through seed maturity(Handley et al., J. Amer. Soc. Hort. Sci. 108:600-605 (1983); Saravitzet al., Plant Physiol. 83:185-189 (1987); Lowell et al., Crop Sci.29:459-465 (1989); Kuo et al., Plant Sci. 125:1-11 (1997), which arehereby incorporated by reference in their entirety). During soybean seeddevelopment in planta, GolS mRNA was first detected in axis tissues at44 days post anthesis (DPA) and in cotyledons at 46 to 48 DPA (Volk,Ph.D. Dissertation, Cornell University, Ithaca, N.Y., pp 176-187 (1998),which is hereby incorporated by reference in its entirety), coincidentwith galactinol accumulation and at the onset of stachyose accumulation(Obendorf et al., Crop Sci. 38:78-84 (1998), which is herebyincorporated by reference in its entirety). GolS transcripts remainedhigh during seed desiccation (Volk, Ph.D. Dissertation, CornellUniversity, Ithaca, N.Y., pp 176-187 (1998), which is herebyincorporated by reference in its entirety). GolS enzyme activity andmRNA increase in response to cold or desiccation (Castillo et al., J.Agric. Food Chem. 38:351-355 (1990); Liu et al., Plant Sci. 134:11-20(1998), which are hereby incorporated by reference in their entirety).Among seven Arabidopsis thaliana GolS genes, three were stressresponsive (Taji et al., Plant J. 29:417-426 (2002), which is herebyincorporated by reference in its entirety). AtGolS1 and AtGolS2 wereinduced by water-deficit stress and high-salinity stress but not by coldstress. AtGolS3 was induced by cold stress but not by drought or saltstress. Soybean seeds matured at 25° C. had increased D-chiro-inositoland fagopyritol B1 compared to seeds matured at 18° C., but galactinolremained unchanged (Obendorf et al., Crop Sci. 38:78-84 (1998), which ishereby incorporated by reference in its entirety), indicating a lack ofresponse to a lower temperature. Similarly, tomato (Lycopersiconesculentum Mill.) seed GolS (LeGolS-1) mRNA increased in maturing seedsbefore desiccation, was concentrated in the radicle tip of mature dryseeds, was induced by desiccation but not cold in germinating seeds, andwas induced by both desiccation and cold in seedling leaves (Downie etal., Plant Physiol. 131:1347-1359 (2003), which is hereby incorporatedby reference in its entirety).

Substrate specificities of soybean GolS and stachyose synthase aredifferent. The lack of soybean GolS activity with D-pinitol, D-ononitol,and sequoyitol as galactosyl acceptors contrasts with the activity ofstachyose synthase with these O-methylated cyclitols (Peterbauer et al.,Plant Physiol. 117:165-172 (1998); Hoch et al., Arch. Biochem. Biophys.366:75-81 (1999); Peterbauer et al., J. Biol. Chem. 277:194-200 (2002),which are hereby incorporated by reference in their entirety). Likewise,activity of GmGolS with D-bornesitol contrasts with the lack of activityof stachyose synthase with D-bornesitol or L-bornesitol (Peterbauer etal., Plant Physiol. 117:165-172 (1998); Hoch et al., Arch. Biochem.Biophys. 366:75-81 (1999), which are hereby incorporated by reference intheir entirety). Lentil (Lens culinaris Medic.) stachyose synthase hasbeen demonstrated to catalyze the biosynthesis of galactopinitols (Hochet al., Arch. Biochem. Biophys. 366:75-81 (1999), which is herebyincorporated by reference in its entirety); this enzyme had low activitywith D-chiro-inositol and no activity with L-chiro-inositol. Bycontrast, adzuki bean (Vigna angularis Ohwi and Ohashi) stachyosesynthase had only a trace of activity with D-pinitol and no activitywith D-chiro-inositol or L-chiro-inositol (Peterbauer et al., PlantPhysiol. 117:165-172 (1998), which is hereby incorporated by referencein its entirety). A recombinant raffinose synthase from pea (Pisumsativum L.) seeds was active with D-ononitol and D-pinitol to formgalactosyl ononitol and galactosyl pinitol using galactinol as thegalactosyl donor (Peterbauer et al., Planta 215:839-846 (2002), which ishereby incorporated by reference in its entirety). This Pisum sativumraffinose synthase also exhibited a neutral α-galactosidase activity(Peterbauer et al., Planta 215:839-846 (2002), which is herebyincorporated by reference in its entirety), consistent with its aminoacid sequence similarity to a family of alkaline α-galactosidases (SeedImbibition Proteins, SIPs) (Carmi et al., Plant J. 33:97-106 (2003),which is hereby incorporated by reference in its entirety). Amulti-functional pea seed stachyose synthase had low activities forbiosynthesis of galactopinitol and verbascose (Peterbauer et al., J.Biol. Chem. 277:194-200 (2002), which is hereby incorporated byreference in its entirety). Collectively, these observations demonstratesubstrate specificity of these multi-functional enzymes to bespecies-specific and product accumulation to be dependent upon theavailability of specific cyclitol substrates to the embryo tissues.Clearly, GmGolS can catalyze the biosynthesis of fagopyritol B1, but notgalactopinitols, in maturing soybean embryos.

Example 4 Biosynthesis of Fagopyritol B1 and Galactopinitols in SoybeanExplants Following Feeding with Free Cyclitols

Soybean is a leguminous plant that bears monocarpic fruit only oncebefore death. During maturation, tissues become yellow starting withradical tips, leaf blades, pod walls, hypocotyls, and cotyledons (Benneret al., Biochemie and Physiologie der Pflanzen 179:269-275 (1984), whichis incorporated herein by reference in its entirety). Yellowing of theseed coat and embryo indicate cessation of dry matter accumulation inthe seed (TeKrony et al., Agronomy Journal 73:553-556 (1981); VerNooy etal., Plant Physiology 82:222-225 (1986), which are hereby incorporatedby reference in their entirety). Leaf yellowing, however, is not alwaysa good indicator of when a given soybean seed has stopped growing(Neumann et al., Plant Physiology 72:182-185 (1983), which isincorporated herein by reference in its entirety). Because there istransport from the leaves to the pod, seed weight may continue toincrease as long as leaves are still alive. Consequently, pod yellowingis the indicator that is often used to determine the time at whichmaximum dry weight is reached (Benner et al., Biochemie and Physiologieder Pflanzen 179:269-275 (1984), which is incorporated herein byreference in its entirety). The onset of this yellowing/desiccation iswhat brings about galactosyl cyclitol accumulation in axis and cotyledontissue (Obendorf et al., Plant Science 132:1-12 (1998); Obendorf et al.,Crop Science 38:78-84 (1998), which are incorporated herein by referencein their entirety).

Soybean seeds accumulate galactosyl cyclitols as opposed to freecyclitols (Horbowicz et al., Seed Science Research 4:385-405 (1994),which is incorporated herein by reference in its entirety). Theseinclude galactosyl derivatives of D-pinitol, D-chiro-inositol, andmyo-inositol in soybean seeds (Obendorf et al., Crop Science 38:78-84(1998), which is incorporated herein by reference in its entirety).Among the fifteen soluble carbohydrates or maturation sugars aresucrose, raffinose and stachyose (raffinose oligosaccharides series),galactopinitol A and galactopinitol B (galactopinitol series), andfagopyritol B1 (fagopyritol series) (Schweizer et al., Carb. Res.95:61-71 (1981); Obendorf et al., Plant Science 132:1-12 (1998);Obendorf et al., Crop Science 38:78-84 (1998), which are incorporatedherein by reference in their entirety). Soluble carbohydrates of thistype may have multiple functions in the desiccation tolerance ofmaturing seeds. They are harmless forms of seed storage products andintracellular osmotic agents contributing to the structural stability oforganelles, membranes, enzymes, proteins, and other macromolecules(Obendorf, Seed Science Research 7:63-74 (1997), which is herebyincorporated by reference in its entirety).

Upon being fed to soybean, free cyclitols undergo biosynthetic reactionsto form galactosyl cyclitols. Several important reactions ofmyo-inositol, D-chiro-inositol, and D-pinitol will be discussedhereafter. Firstly, myo-inositol is encountered in all living cells andis the primary source for the biosynthesis of various cyclitols. Feedingmyo-inositol to soybean promotes the production of galactinol. The threecomponents of the galactinol series are myo-inositol, galactinol, anddigalactosyl myo-inositol. Galactinol is far-reaching in its ability todonate galactose for the formation of stachyose, raffinose, andverbascose (Peterbauer et al., Seed Science Research 11:185-198 (2001);Taji et al., Plant Journal 29:417-426 (2002), which are incorporatedherein by reference in their entirety). If galactose is donated toanother galactinol molecule, digalactosyl myo-inositol is formed.Secondly, feeding D-pinitol enhances accumulation of galactopinitol Aand galactopinitol B common in legume seeds (Odorcic et al., The Biologyof Seeds: Recent Research Advances. Wallingford, UK, CABI Publishing(2003), which is incorporated herein by reference in its entirety). Asstachyose accumulates during soybean seed maturation, galactopinitolsalso increase (Obendorf et al., Crop Science 38:78-84 (1998), which isincorporated herein by reference in its entirety). In addition to this,galactopinitols accumulate during precocious maturation of immatureseeds. Lastly, feeding of D-chiro-inositol results in enhancedaccumulation of fagopyritol B1 (Odorcic et al., The Biology of Seeds:Recent Research Advances. Wallingford, UK, CABI Publishing (2003), whichis incorporated herein by reference in its entirety). The fagopyritol Bseries enhanced through feeding consists of fagopyritol B1 (firstidentified in soybean seeds), D-chiro-inositol, fagopyritol B2, andfagopyritol B3, which accumulate in buckwheat seeds (Obendorf, SeedScience Research 7:63-74 (1997); Horbowicz et al., Planta 205:1-11(1998), which are incorporated herein by reference in their entirety). Anovel series of fagopyritols, fagopyritol A1, fagopyritol A2, andfagopyritol A3, also accumulate in buckwheat seeds (Horbowicz et al.,Planta 205:1-11 (1998); Obendorf et al., Carbohydrate Research328:623-627 (2000); Steadman et al., Carbohydrate Research 331:19-25(2001), which are incorporated herein by reference in their entirety).

Knowledge of the translocation patterns of cyclitols is indispensable inunderstanding their function (Noodén et al., Journal of Plant GrowthRegulation 2:265-279 (1984), which is incorporated herein by referencein its entirety). Previous studies used labeled chemicals, hormones, orsugars in order to observe these very translocation patterns withinplants of interest. In an experiment done by Noodén and Letham in 1983,for example, ³H (ring-labeled) zeatin riboside was used to trace theproduction of the hormone cytokinin. The hormone was fed to soybeanexplants and transported via the transpiration stream. This biologicalmarker allowed for the clear observation of transport from the xylem tothe leaf and embryo of the explant. This experiment also resulted inleaves retaining their green color longer, which is important inexperimentation with soybean explants (Noodén et al., Journal of PlantGrowth Regulation 2:265-279 (1984), which is incorporated herein byreference in its entirety). A previous study by Quebedeaux and Chollet(Quebedeaux et al., Plant Physiology 55:745-748 (1975), which is herebyincorporated by reference in its entirety) used radioactive tracers todemonstrate that the pods (and seeds contained therein) of soybeans arethe main sinks for the photosynthetic assimilates from the leaf,indicating that the decrease in the production of photosynthate istherefore due to the decrease in photosynthetic activity of the plant,which accompanies senescence (Benner et al., Biochemie and Physiologicder Pflanzen 179:269-275 (1984), which is incorporated herein byreference in its entirety). In addition to these methods, translocationpatterns can also be observed through analysis of products formedfollowing exogenous feeding of large quantities of the compound(s) ofinterest.

It is known that myo-inositol is biosynthesized in soybean embryos.Johnson and Wang (Johnson et al., J. Biol. Chem. 271:17215-17218 (1996),which is hereby incorporated by reference in its entirety) demonstratedthat 1L-myo-inositol 1-phosphate synthase (also known as 1D-myo-inositol3-phosphate synthase, MIPS) catalyzes the transformation of Glc-6-P to1L-myo-inositol 1-phosphate in embryos of developing legume seeds.However, it remains unknown whether D-pinitol or D-chiro-inositol arebiosynthesized in the embryo. In order to understand the function ofcyclitols, it is necessary to first understand how they are transportedand from where they are transported. Therefore, one objective of thisExample was to determine which cyclitols are biosynthesized in soybeanembryos and which are transported to the embryo from the leaves.

Several studies provide evidence in support of the hypothesis thatD-pinitol and D-chiro-inositol are biosynthesized in the leaves ofsoybean plants. Labeling studies done by Diettrich and Brandi (Diettrichet al., Phytochemistry 26:1925-1926 (1987), which is hereby incorporatedby reference in its entirety), for example, showed that myo-inositolgoes to D-ononitol (FIG. 20, reaction d) and afterwards to D-pinitol(FIG. 20, reaction e, f), and then presumably to D-chiro-inositol (FIG.20, reaction g) in legume leaves. Kuo (Kuo et al., Phytochemistry45:29-35 (1997), which is hereby incorporated by reference in itsentirety) demonstrated that the concentration of D-pinitol was highestin seed coats and lower in axis and cotyledon tissues, suggesting thatD-pinitol is biosynthesized in maternal tissue and transported tosoybean embryos. In addition to this, soybean and alfalfa (Medicagosativa L.) somatic embryos also appear to be deficient in D-pinitol andgalactopinitols (Horbowicz et al., Plant Science 109:191-198 (1995);Obendorf et al., Mol. Cell. Biol. Soybean 6:40 (1996); Chanprame et al.,in Vitro Cell Developmental Biology—Plant 34:64-68 (1998), which areincorporated herein by reference in their entirety), and total D-pinitolor total D-chiro-inositol in soybean zygotic embryos matured in vitrodid not exceed that present in embryos before culture (Obendorf et al.,Plant Science 132:1-12 (1998); Obendorf et al., Crop Science 38:78-84(1998), which are incorporated herein by reference in their entirety),indicating a lack of D-pinitol and D-chiro-inositol biosynthesis byembryo tissues. myo-inositol 6-O-methyltransferase (mI6OMT or IMT,S-adenosyl-L-methionine:myo-inositol O-methyltransferase, EC 2.1.1.129)that forms D-ononitol, is located in leaves and stems (Wanek et al.,Physiologia Plantarum 101:416-424 (1997); Streeter et al., Plant, Celland Environment 24:429-438 (2001), which are incorporated herein byreference in their entirety). Soybean somatic embryos transformed with agene for this enzyme form D-ononitol but not D-pinitol indicating thatsoybean somatic embryos do not express the enzymes that form D-pinitol.Soybean leaves accumulate mostly D-pinitol with small amounts ofD-chiro-inositol, myo-inositol and D-ononitol (Streeter, Crop Sci.41:1985-1987 (2001), which is hereby incorporated by reference in itsentirety). Using this background information in conjunction with theknowledge that D-pinitol is a proposed precursor to D-chiro-inositol, itwas hypothesized that though myo-inositol is biosynthesized in soybeanembryos, D-chiro-inositol and D-pinitol are biosynthesized in the leavesand afterwards transported to the seeds. If this hypothesis is correct,then increasing the concentration of D-pinitol and D-chiro-inositol insoybean explants via exogenous feeding should result in a dramaticincrease in the accumulation of fagopyritol B1 and galactopinitols inthe embryo. However, if D-pinitol and D-chiro-inositol arebiosynthesized in the embryo, then exogenous feeding of free cyclitolsshould have a less pronounced effect on galactosyl cyclitolconcentrations in the seed.

Materials and Methods

Soybean plants [Glycine max (L.) Merrill cv. Chippewa 64] were grown ina greenhouse at 27° C. days (14 hours) and 22° C. nights (10 hours) withnatural light supplemented by 640-μmol m⁻² s⁻¹ artificial light fromSylvania 1000-watt metal halide lamps.

Plants were excised above the third node from the bottom and below thethird node from the top before leaf senescence was evident as was doneby Neumann et al. Plant Physiology 72:182-185 (1983), which is herebyincorporated by reference in its entirety. Explants were cut mid podfill(about 35 days after flowering), when the pods were still green andapproximately 7.2 mm in width, and the seeds weighed about 250 mg freshweight. Pod number was reduced to one, containing three seeds. Eachexplant included one node, one leaf, one pod, and one internode. The cutbasal end of the internode (stem) of the explants was placed in 50 mMsolutions of cyclitols: 50 mM myo-inositol, 50 mM D-pinitol, 50 mMD-chiro-inositol, and a control without cyclitols, all in 1% sucrose byweight, and all containing 10 mM asparagine and kinetin, a cytokinin.These solutions were loaded into the explant through the cut stem andtransported to the leaf by the transpiration stream and to the embryothrough the phloem. A fourth solution consisting of 10 mM asparagine andkinetin in 1% sucrose (by weight) served as the control. Solutions werefed to explants for one week, and explants were allowed to dry, afterwhich seeds were moved to the desiccators and fully dried (to 6%moisture) during a period of 14 days at 12% relative humidity over asaturated solution of LiCl.

After the seeds had slow dried, extraction and analysis of solublecarbohydrates was performed. Cotyledon and axis tissues were separated,weighed, pulverized in liquid nitrogen with a mortar and pestle, andhomogenized in a ground glass homogenizer with 2.2 ml of ethanol:water(1:1, v/v), containing 300 μg (cots) or 100 μg (axis) of phenylα-D-glucoside as the internal standard, heated at 80° C. for 45 minutes,and centrifuged at 27,000×g for 20 minutes. Clear supernatants werepassed through a 10,000 MW cutoff filter and evaporated to dryness withnitrogen gas. Residues were stored overnight in a desiccator with P₂O₅to remove traces of water and afterwards derivatized withtrimethylsilylimidazole:pyridine (1:1, v/v). Analysis of solublecarbohydrates was done using a Hewlett Packard 5890 Series II gaschromatograph equipped with a flame ionization detector and ChemStationsoftware as previously described (Horbowicz et al., Seed ScienceResearch 4:385-405 (1994); Obendorf et al., Crop Science 38:78-84(1998), which are incorporated herein by reference in their entirety).The amounts of each soluble carbohydrate present in the samples wasdetermined by regression equations calculated from gas chromatograms ofknown standards, allowing the relative amounts of cyclitols present inthe leaf and embryo as a result of the feeding of excess cyclitols to bedetermined. Soluble carbohydrate composition is reported as mean±SE ofthe mean on a dry weight basis for six replicate samples of cotyledonsfrom mature seeds.

Results

Overall, none of the feeding experiments resulted in large changes insucrose, raffinose, or stachyose except for some low values observed inexplants fed with D-chiro-inositol. Results for the experiments wereconsistent with the results and interpretations of feeding experimentswhere cyclitols were fed to immature soybean embryos (Odorcic et al.,The Biology of Seeds: Recent Research Advances. Wallingford, UK, CABIPublishing (2003), which is incorporated herein by reference in itsentirety).

Myo-inositol

Feeding 50 mM myo-inositol to soybean explants slightly increased freemyo-inositol and caused a 50% increase in galactinol in axis andcotyledon tissue (Tables 1 and 2).

TABLE 1 Concentration of soluble carbohydrates in cotyledons of maturesoybean seeds after feeding explants 50 mM myo-inositol,D-chiro-inositol, or D-pinitol. A B C D myo-Inositol D-chiro-InositolD-Pinitol Control D-Pinitol 7.77 ± 0.92 6.00 ± 0.52 35.77 ± 2.50  8.34 ±1.12 Galactopinitol A 2.01 ± 0.16 1.89 ± 0.14 5.81 ± 0.38 1.59 ± 0.15Galactopinitol B 1.76 ± 0.21 1.62 ± 0.18 4.88 ± 3.10 1.60 ± 0.22Ciceritol 0.63 ± 0.08 0.34 ± 0.11 1.13 ± 0.16 0.85 ± 0.07D-chiro-Inositol 5.15 ± 0.77 15.59 ± 2.08  1.63 ± 0.11 1.63 ± 0.26Fagopyritol B1 1.78 ± 0.23 21.11 ± 2.06  1.77 ± 0.11 1.05 ± 0.08Fagopyritol B2 0.25 ± 0.07 1.52 ± 0.47 0.16 ± 0.02 0.15 ± 0.04Myo-Inositol 2.35 ± 0.79 0.58 ± 0.05 0.67 ± 0.07 1.69 ± 0.44 Galactinol0.35 ± 0.06 0.23 ± 0.05 0.05 ± 0.01 0.25 ± 0.04 Sucrose 37.73 ± 4.81 27.88 ± 4.09  32.49 ± 2.05  48.76 ± 7.62  Raffinose 11.22 ± 1.12  7.18 ±0.58 9.73 ± 0.46 11.00 ± 1.47  Stachyose 23.10 ± 1.94  12.63 ± 1.40 14.60 ± 1.06  24.51 ± 3.73 

TABLE 2 Concentration of soluble carbohydrates in axis of mature soybeanseeds after feeding explants 50 mM myo-inositol, D-chiro-inositol, orD-pinitol. A B C D myo-Inositol D-chiro-Inositol D-Pinitol ControlD-Pinitol 3.65 ± 0.03 4.07 ± 0.38 21.40 ± 2.41  4.51 ± 0.58Galactopinitol A 4.61 ± 0.41 4.96 ± 0.30 12.14 ± 1.06  3.65 ± 0.35Galactopinitol B 3.17 ± 0.40 3.61 ± 0.30 9.42 ± 0.88 2.84 ± 0.42Ciceritol 0.65 ± 0.12 0.37 ± 0.15 1.58 ± 0.26 0.79 ± 0.23D-chiro-Inositol 1.08 ± 0.14 12.31 ± 1.44  1.28 ± 0.30 0.61 ± 0.12Fagopyritol B1 2.81 ± 0.28 30.95 ± 2.46  2.99 ± 0.22 1.79 ± 0.22Fagopyritol B2 0.24 ± 0.09 1.62 ± 0.50 0.08 ± 0.02 0.14 ± 0.06Myo-Inositol 1.52 ± 0.15 1.37 ± 0.13 0.98 ± 0.13 1.19 ± 0.20 Galactinol0.89 ± 0.10 1.00 ± 0.07 0.58 ± 0.08 0.69 ± 0.08 Sucrose 34.02 ± 3.52 32.18 ± 4.31  30.86 ± 4.11  35.59 ± 5.63  Raffinose 8.87 ± 0.77 6.61 ±0.81 9.81 ± 1.08 1.030 ± 0.76  Stachyose 24.56 ± 2.55  17.95 ± 1.79 23.16 ± 2.50  22.02 ± 2.32 No significant changes in the amount of stachyose, raffinose, D-pinitol,or galactopinitols were observed. A 3.15-fold increase in freeD-chiro-inositol was also observed in cotyledons and D-chiro-inositolconcentrations were doubled in axis tissue. Still, there was nosignificant increase in concentrations of fagopyritol B1.D-Chiro-inositol

Feeding 50 mM D-chiro-inositol to soybean explants caused a 9.6-foldincrease in free D-chiro-inositol, a 20-fold increase in fagopyritol B1,and a 10-fold increase in fagopyritol B2 in cotyledon tissues (Table 1).Free myo-inositol decreased but galactinol in cotyledons remainedunchanged. Feeding D-chiro-inositol to soybean explants also resulted ina 20-fold increase in free D-chiro-inositol in axis tissues (Table 2).This corresponded with a 17-fold increase in fagopyritol B1 and an11-fold increase in fagopyritol B2. All of the D-chiro-inositol fedexplants had shriveled seeds, while those explants that were fedmyo-inositol, D-pinitol, or the control treatment, had full and roundseeds.

D-pinitol

Feeding D-pinitol quadrupled free D-pinitol and tripled galactopinitolsin both axis and cotyledon tissues (Tables 1 and 2). Ciceritolconcentrations increased 30% in cotyledon tissue and they doubled inaxis tissue. myo-Inositol and galactinol were decreased 25%, and freeD-chiro-inositol concentrations in axes doubled.

Discussion

Relative amounts of soluble carbohydrates observed can be attributed tobiochemical pathways in soybean and the roles that D-pinitol,D-chiro-inositol, and myo-inositol play in these pathways.

In soybean explants, galactinol synthase (GolS or GAS) producesgalactinol from myo-inositol and UDP-galactose (FIG. 21). Galactinolthen undergoes two reactions. In the first reaction, galactinol acts asa galactosyl donor to sucrose, which reacts with raffinose synthase(RFS) to produce raffinose and myo-inositol as a by-product. Raffinoseand galactinol then reacts with stachyose synthase (STS) to producestachyose and myo-inositol as a by-product. In the second reaction,galactinol and D-pinitol react with STS to produce galactopinitol A andgalactopinitol B. Subsequent reactions with STS produce ciceritol (adigalactosyl pinitol A) from galactinol and galactopinitol A, anddigalactosyl pinitol B from galactinol and galactopinitol B (FIG. 21).

When feeding 50 mM myo-inositol, there were high levels of galactinol,the galactosyl donor for galactopinitol biosynthesis, present. The lackof increase in accumulation of galactopinitols may have been due tolimited levels of D-pinitol in the explant. Biosynthesis ofD-chiro-inositol in legumes is believed to be via myo-inositol toD-ononitol to D-pinitol to D-chiro-inositol (FIG. 20, reactions d, e, f,g; Dittrich et al., Phytochemistry 26:1925-1926 (1987) which is herebyincorporated by reference in its entirety). If the D-pinitol levels werelow, it follows that D-chiro-inositol should also have been low, butthis was not the case. High levels of D-chiro-inositol in the cotyledonssuggest that myo-inositol, rather than D-pinitol, is a direct precursorto production of D-chiro-inositol in the leaves (FIG. 20, reactions i,j). In the absence of D-pinitol, myo-inositol goes to D-myo-1-inosose,and then to D-chiro-inositol (FIG. 20, reactions i, j). The high levelsof myo-inositol present in the soybean explant following feeding withexogenous myo-inositol may limit the accumulation of raffinose andstachyose by feedback inhibition in the cotyledons of the seed. Sincemyo-inositol is produced as a byproduct, exogenous myo-inositoldecreased the progress of the reaction of sucrose and galactinol by RFS,explaining why raffinose and stachyose levels stayed the same with thistreatment.

Soybean galactinol synthase (GmGolS or GAS) produces fagopyritol B1 fromD-chiro-inositol and UDP-galactose (FIG. 22). When feeding 50 mMD-chiro-inositol to soybean explants, a decrease in myo-inositol andD-pinitol was observed. Because myo-inositol and D-pinitol areprecursors to D-chiro-inositol (FIG. 20, reactions d, e, f, g; Dittrichet al., Phytochemistry 26:1925-1926 (1987), which is hereby incorporatedby reference in its entirety), they may not have been needed to produceD-chiro-inositol because it was fed to the explant in excess. The reasonfor decreased production of raffinose and stachyose is unknown.Decreases in galactosyl and digalactosyl pinitols are due to decreasesin their precursor, D-pinitol. Increased levels of D-chiro-inositolcaused fagopyritol B1 and fagopyritol B2 to increase as expected.

In this experiment, feeding D-pinitol increased levels of free D-pinitolin the seed. This increase served to increase the amount of galactosylpinitols and, after reaction with STS, production of digalactosylpinitol B. High levels of D-pinitol may have also temporarily increasedD-chiro-inositol levels (FIG. 20, reaction g), which subsequently wenttowards increasing fagopyritol B1 production. The increased level ofdigalactosyl myo-inositol accounts for the decreased levels ofgalactinol and myo-inositol.

myo-Inositol is biosynthesized in embryo tissues of developing legumeseeds (Johnson et al., Journal of Biological Chemistry 271, 17215-17218(1996); Hegeman et al., Plant Physiology 125:1941-1948 (2001); Hitz etal., Plant Physiology 128:650-660 (2002), which are incorporated byreference in their entirety). D-Pinitol is biosynthesized in leaves frommyo-inositol through D-ononitol as precursor (FIG. 20, reactions d, e,f; Dittrich et al., Phytochemistry, 26:1925-1926 (1987), which is herebyincorporated by reference in its entirety) and D-chiro-inositol isbelieved to be biosynthesized by demethylation of D-pinitol (FIG. 20,reaction g; see review by Obendorf, Seed Sci. Res. 7:63-74 (1997), whichis hereby incorporated by reference in its entirety). It is not known ifD-pinitol and D-chiro-inositol are biosynthesized in cotyledons ofseeds. Further, the enzymes and genes responsible for the biosynthesisof D-pinitol (FIG. 20, reactions e, f) and D-chiro-inositol (FIG. 20,reaction g or FIG. 20, reactions i, j) are unknown (Obendorf, SeedScience Research 7:63-74 (1997), which is hereby incorporated byreference in its entirety). The results herein are consistent with theinterpretation that both of D-pinitol and D-chiro-inositol arebiosynthesized in leaves and transported to seeds. Of special interestis the evidence presented herein that D-chiro-inositol may bebiosynthesized directly from myo-inositol, either instead of or inaddition to demethylation of D-pinitol.

The results in this Example are consistent with the followinginterpretations: myo-inositol is formed in maternal tissues and inembryos of seeds, D-pinitol and D-chiro-inositol are biosynthesized inmaternal tissues (leaves) and transported to seeds, D-chiro-inositol maybe biosynthesized directly from myo-inositol, galactinol synthaseutilizes D-chiro-inositol to form fagopyritol B1, stachyose synthaseutilizes D-pinitol to form galactopinitols, and feeding free cyclitolsto soybean explants does not increase raffinose and stachyoseaccumulation in cotyledons of soybean seeds.

Example 5 Soybean Explant Feeding Experiments

Soybean explants, consisting of a stem segment with attached leaf andpod, were cultured as the soybean explants described in Example 4. Inthis example, the soybean explant system was used to study the timing oftransport of cyclitols, fed through the stem, to the developing soybeanseed and the timing of their incorporation into galactosyl cyclitols inaxis, cotyledons, and seed coat of developing and maturing soybeanseeds. myo-inositol, D-pinitol and D-chiro-inositol were fed to soybeanexplants as described in Example 4, except that 50 mM cyclitol in 1%sucrose solution was fed to stems of soybean explants for three daysfollowed by slow drying. Soluble carbohydrates were extracted andassayed by high resolution gas chromatography after slow drying of seeds(as described in Example 4).

TABLE 3 Accumulation of soluble carbohydrates in soybean axis (μg/axis)after 3 days transport of sucrose (1% solution) and myo-inositol (50 mM)into the stem of soybean explants and after slow drying of seeds for 2,4, or 14 days (micrograms/1 axis) Before After 2 days After 4 days After14 days Soluble slow drying slow drying slow drying slow dryingCarbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 D-Pinitol5.89 5.97 5.05 5.72 2.74 2.86 6.70 5.72 Unknown 0.91 0.67 0.57 0.49 0.520.38 1.25 1.46 D-chiro-Inositol 1.98 2.17 3.65 4.69 3.40 1.75 9.39 5.12myo-Inositol 37.08 37.50 3.34 1.96 1.74 1.69 4.22 3.84 Sucrose 143.88139.88 38.38 76.58 50.48 82.93 77.01 63.96 Galactopinitol A 4.31 0 5.868.20 9.93 11.97 13.99 10.23 Galactopinitol B 0.72 0 1.29 2.20 3.06 4.605.50 3.91 Fagopyritol B1 0 0 3.15 5.91 6.25 7.43 11.14 6.13 Galactinol3.01 3.59 12.80 10.83 7.31 4.07 6.76 4.14 Raffinose Tr 0 5.44 1.83 6.377.57 14.51 11.98 Ciceritol Tr 0 0 0 0.39 0.42 0.56 0.29 Fagopyritol B2 00 0 0 0 0 0 0 Stachyose 0 0 9.92 6.33 62.18 55.60 89.46 57.18

TABLE 4 Accumulation of soluble carbohydrates in soybean axis (μg/axis)after 3 days transport of sucrose (1% solution) and D-chiro-inositol (50mM) into the stem of soybean explants and after slow drying of seeds for2, 4, or 14 days (micrograms/1 axis) Before After 2 days After 4 daysAfter 14 days Soluble slow drying slow drying slow drying slow dryingCarbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 D-Pinitol6.94 6.11 6.18 7.87 6.21 3.86 7.68 4.13 Unknown 0.47 0 0.39 0.46 0.650.42 0 0.92 D-chiro-Inositol 25.26 22.53 20.29 25.33 11.74 7.67 39.9926.32 myo-Inositol 18.22 16.89 1.93 2.75 1.27 1.03 0.92 1.01 Sucrose111.55 149.75 22.44 28.32 35.75 23.35 59.50 30.63 Galactopinitol A 0 06.35 6.24 13.54 12.18 15.50 10.18 Galactopinitol B 0 0 1.47 1.56 6.354.73 9.35 5.03 Fagopyritol B1 2.04 0 32.08 25.93 81.94 73.53 78.96 55.76Galactinol 0 2.71 6.29 10.19 3.46 2.25 4.23 2.27 Raffinose 0.23 0 2.573.37 2.61 3.24 6.68 5.73 Ciceritol 0 0 0 0 0 0.27 0 2.69 Fagopyritol B20 0 0 0 0 1.72 2.07 0 Stachyose 0 0 11.56 8.94 49.55 22.07 47.82 39.47

TABLE 5 Accumulation of soluble carbohydrates in soybean axis (μg/axis)after 3 days transport of sucrose (1% solution) and D-pinitol (50 mM)into the stem of soybean explants and after slow drying of seeds for 2,4, or 14 days (micrograms/1 axis) Before After 2 days After 4 days After14 days Soluble slow drying slow drying slow drying slow dryingCarbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 D-Pinitol16.89 18.34 22.93 20.86 14.00 20.05 26.07 56.18 Unknown 1.30 1.47 0 00.69 0.45 0.95 1.53 D-chiro-Inositol 1.30 2.20 2.40 1.39 0.59 0.48 1.831.32 myo-Inositol 13.58 15.43 1.58 2.01 1.12 0 2.22 3.62 Sucrose 130.10160.58 32.16 37.53 66.76 27.38 48.95 56.62 Galactopinitol A 0 0 7.629.41 25.04 19.32 24.72 32.49 Galactopinitol B 0 0 3.09 3.49 9.62 7.2510.61 12.71 Fagopyritol B1 0 0 4.30 2.75 5.79 4.01 6.41 8.76 Galactinol2.27 2.97 6.40 10.37 5.46 2.87 3.92 4.18 Raffinose 0 0 6.33 3.86 7.493.46 7.60 11.19 Ciceritol 0 0 0 0.80 0.89 0.56 1.42 1.43 Fagopyritol B20 0 0 0 0 0 0 1.26 Stachyose 0 0 11.91 7.69 79.54 28.64 54.54 78.56

TABLE 6 Accumulation of soluble carbohydrates in soybean axis (μg/axis)after 3 days transport of sucrose (1% solution) without cyclitols intothe stem of soybean explants and after slow drying of seeds for 2, 4, or14 days (micrograms/1 axis) Before After 2 days After 4 days After 14days Soluble slow drying slow drying slow drying slow dryingCarbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 D-Pinitol10.61 11.12 7.28 10.54 4.24 4.89 8.64 4.32 Unknown 0.32 0.98 0.85 0.480.69 0.41 1.58 1.68 D-chiro-Inositol 1.96 2.03 0.89 0.94 0.39 0.30 1.920.62 myo-Inositol 16.11 20.11 2.42 2.14 1.49 1.66 2.62 2.36 Sucrose230.72 282.09 48.81 84.07 87.13 159.22 115.27 91.58 Galactopinitol A 0 07.55 9.97 17.42 16.07 16.14 12.28 Galactopinitol B 0 0 1.81 2.60 7.117.06 7.78 5.09 Fagopyritol B1 1.70 0 3.51 5.52 7.09 6.40 8.41 4.98Galactinol 1.20 5.49 11.09 13.41 5.60 4.29 4.38 4.27 Raffinose 0.62 2.655.83 8.95 8.58 12.44 13.14 8.02 Ciceritol 0 0 0.01 0.07 0.80 0.71 0.940.63 Fagopyritol B2 0 0 5.67 0 0 0 0 0 Stachyose 0 7.83 0 27.73 96.03118.05 101.39 69.71

TABLE 7 Accumulation of soluble carbohydrates in soybean cotyledons(μg/cotyledon) after 3 days transport of sucrose (1% solution) andmyo-inositol (50 mM) into the stem of soybean explants and after slowdrying of seeds for 2, 4, or 14 days (micrograms/1 cot) Before After 2days After 4 days After 14 days Soluble slow drying slow drying slowdrying slow drying Carbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep1 Rep 2 D-Pinitol 99.88 127.21 121.52 132.23 101.97 128.68 61.67 95.93Unknown 4.53 8.94 53.99 63.64 86.25 85.24 62.60 111.06 D-chiro-Inositol52.20 55.90 133.52 129.44 150.98 192.49 95.36 137.86 myo-Inositol 257.87275.47 67.54 29.93 21.34 12.69 7.82 11.96 Sucrose 1429.70 1496.60 635.91995.62 921.47 1952.20 717.30 1228.20 Galactopinitol A 9.46 10.00 12.8410.98 28.27 54.91 34.57 28.69 Galactopinitol B 0 0 6.27 3.19 11.92 28.1115.29 9.70 Fagopyritol B1 0 0 7.36 14.06 29.15 86.71 50.31 49.29Galactinol 0 0 85.06 104.07 57.03 25.34 14.65 14.10 Raffinose 0 0 29.2089.65 122.10 351.33 165.12 218.44 Ciceritol 0 0 0 0 0 0 2.01 0Fagopyritol B2 0 0 0 0 0 0 1.29 5.27 Stachyose 0 0 0 50.41 224.95 774.04399.25 323.00

TABLE 8 Accumulation of soluble carbohydrates in soybean cotyledon(μg/cotyledon) after 3 days transport of sucrose (1% solution) andD-chiro-inositol (50 mM) into the stem of soybean explants and afterslow drying of seeds for 2, 4, or 14 days (micrograms/1 cot) BeforeAfter 2 days After 4 days After 14 days Soluble slow drying slow dryingslow drying slow drying Carbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2Rep 1 Rep 2 D-Pinitol 175.37 149.51 140.00 177.82 131.32 171.09 91.5090.76 Unknown 49.44 36.70 65.35 59.28 74.11 69.17 39.34 56.75D-chiro-Inositol 518.30 476.04 635.01 719.52 388.25 589.84 335.21 286.00myo-Inositol 138.61 142.47 21.83 55.39 4.25 10.39 5.77 3.80 Sucrose1978.90 2102.30 360.00 363.10 395.07 579.04 580.31 736.23 GalactopinitolA 15.98 0 12.41 16.08 47.20 42.69 48.41 47.44 Galactopinitol B 0 0 0 022.34 20.06 25.58 23.82 Fagopyritol B1 10.55 0 45.25 25.67 711.00 535.23612.60 626.65 Galactinol 14.29 0 42.99 42.35 15.89 32.53 11.02 9.34Raffinose 7.28 10.73 21.08 15.23 122.16 124.39 198.46 211.32 Ciceritol 06.06 13.35 0 12.69 0 3.82 2.83 Fagopyritol B2 0 0 0 4.57 12.10 11.1223.98 22.70 Stachyose 0 0 44.86 0 308.62 277.15 357.02 349.60

TABLE 9 Accumulation of soluble carbohydrates in soybean cotyledon(μg/cotyledon) after 3 days transport of sucrose (1% solution) andD-pinitol (50 mM) into the stem of soybean explants and after slowdrying of seeds for 2, 4, or 14 days (micrograms/1 cot) Before After 2days After 4 days After 14 days Soluble slow drying slow drying slowdrying slow drying Carbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep1 Rep 2 D-Pinitol 351.23 497.66 428.40 431.73 472.26 438.53 160.89303.14 Unknown 62.38 62.50 46.56 38.08 76.63 85.85 24.57 45.01D-chiro-Inositol 33.59 27.90 46.09 46.18 25.69 68.75 14.91 32.16myo-Inositol 116.75 167.27 20.25 28.65 2.77 7.67 4.02 6.03 Sucrose1364.90 2193.00 322.30 431.68 463.59 849.80 369.28 614.41 GalactopinitolA 9.09 9.60 12.29 8.49 94.29 117.43 86.03 104.22 Galactopinitol B 0 0 01.48 41.39 50.76 29.37 40.39 Fagopyritol B1 0 3.39 4.99 4.57 51.96 69.7629.66 51.85 Galactinol 0 0 28.42 40.81 20.87 37.07 11.23 10.39 Raffinose0 4.09 10.85 15.22 72.18 187.91 77.60 162.90 Ciceritol 0 0 0 2.47 0 3.324.88 4.85 Fagopyritol B2 0 0 4.91 0 15.04 11.92 1.77 0 Stachyose 0 0 0 0211.21 552.89 268.51 427.70

TABLE 10 Accumulation of soluble carbohydrates in soybean cotyledon(μg/cotyledon) after 3 days transport of sucrose (1% solution) withoutcyclitols into the stem of soybean explants and after slow drying ofseeds for 2, 4, or 14 days (micrograms/1 cot) Before After 2 days After4 days After 14 days Soluble slow drying slow drying slow drying slowdrying Carbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2D-Pinitol 199.64 176.34 162.30 222.80 187.65 166.14 98.90 153.50 Unknown11.90 12.07 22.73 20.87 60.13 5840 14.10 8.01 D-chiro-Inositol 45.5737.40 50.36 57.42 69.00 73.60 54.07 46.46 myo-Inositol 127.35 113.6623.50 29.70 12.71 15.03 7.16 16.01 Sucrose 2188.80 2395.60 731.21 923.691035.10 2211.00 1281.70 1787.10 Galactopinitol A 9.90 9.21 9.17 9.30114.04 84.24 102.80 68.60 Galactopinitol B 4.55 1.73 1.56 1.54 40.6736.28 49.91 30.24 Fagopyritol B1 1.89 2.10 4.72 13.12 72.21 62.04 83.9357.10 Galactinol 1.88 2.16 83.71 166.27 52.22 25.06 28.60 26.57Raffinose 4.97 2.41 34.55 85.98 200.22 312.69 273.49 444.34 Ciceritol 00 1.63 0 1.79 2.22 10.22 4.63 Fagopyritol B2 0 0 2.69 0 11.94 2.08 4.524.65 Stachyose 0 0 12.20 18.15 773.51 516.66 1331.00 904.20

TABLE 11 Accumulation of soluble carbohydrates in soybean seed coats(μg/seed coat) after 3 days transport of sucrose (1% solution) andmyo-inositol (50 mM) into stem of soybean explants and after slow dryingof seeds for 2, 4, or 14 days (micrograms/1 seed coat) After 2 daysAfter 4 days After 14 days Soluble slow drying slow drying slow dryingCarbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 D-Pinitol 11.24 9.356.81 12.13 15.41 D-chiro-Inositol 15.00 17.15 7.20 13.32 22.63myo-Inositol 38.71 30.23 4.83 6.94 9.10 Sucrose 21.35 23.43 14.28 78.62138.83 Galactopinitol A 0 0 4.87 6.98 7.08 Galactopinitol B 0 0 1.07 02.22 Fagopyritol B1 3.04 1.97 1.90 5.51 6.47 Galactinol 0 0 0 2.61 2.91Raffinose 1.58 0 0 7.44 24.24 Ciceritol 0 0 0 0 0 Fagopyritol B2 0 0 0 00 Stachyose 0 0 0 20.21 35.12

TABLE 12 Accumulation of soluble carbohydrates in soybean seed coat(μg/seed coat) after 3 days transport of sucrose (1% solution) andD-chiro- inositol (50 mM) into the stem of soybean explants and afterslow drying of seeds for 2, 4, or 14 days (micrograms/1 seed coat) After2 days After 4 days After 14 days Soluble slow drying slow drying slowdrying Carbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 D-Pinitol 6.555.97 14.69 10.95 12.79 16.30 D-chiro-Inositol 193.33 173.15 169.87149.60 87.18 134.18 myo-Inositol 12.32 11.96 5.53 5.38 3.76 4.51 Sucrose16.60 17.20 2.65 2.33 9.81 59.33 Galactopinitol A 0 4.13 4.72 4.29 07.33 Galactopinitol B 0 0.79 1.76 0.78 0 3.14 Fagopyritol B1 3.52 4.889.14 8.69 14.54 51.81 Galactinol 0 1.30 0 1.19 0 1.60 Raffinose 0 0 0.360.18 0 10.08 Ciceritol 0 0 0 0 0 0 Fagopyritol B2 0 0 0 0 0 1.35Stachyose 0 0 0 0 0 15.45

TABLE 13 Accumulation of soluble carbohydrates in soybean seed coat(μg/seed coat) after 3 days transport of sucrose (1% solution) andD-pinitol (50 mM) into the stem of soybean explants and after slowdrying of seeds for 2, 4, or 14 days (micrograms/1 seed coat) After 2days After 4 days After 14 days Soluble slow drying slow drying slowdrying Carbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 D-Pinitol 78.0673.32 58.50 90.19 50.78 96.60 D-chiro-Inositol 4.66 4.29 4.05 5.81 4.528.11 myo-Inositol 10.61 14.11 2.68 2.62 4.13 3.58 Sucrose 15.56 23.402.73 4.47 33.23 78.92 Galactopinitol A 0 5.55 4.81 5.72 10.65 19.70Galactopinitol B 0 3.09 1.44 2.10 3.57 6.98 Fagopyritol B1 0 2.10 1.661.66 4.07 10.12 Galactinol 0 0 0 0 0 2.77 Raffinose 0 0 0 0.54 0 18.53Ciceritol 0 0 0 0 0 0 Fagopyritol B2 0 0 0 0 0 0 Stachyose 0 0 0 0 064.19

TABLE 14 Accumulation of soluble carbohydrates in soybean seed coats(μg/seed coat) after 3 days transport of sucrose (1% solution) withoutcyclitols into the stem of soybean explants and after slow drying ofseeds for 2, 4, or 14 days (micrograms/1 seed coat) After 2 days After 4days After 14 days Soluble slow drying slow drying slow dryingCarbohydrate Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 D-Pinitol 16.22 12.1520.16 19.36 25.66 16.34 D-chiro-Inositol 3.27 3.10 3.77 1.74 3.38 2.68myo-Inositol 12.27 12.84 5.67 5.65 3.88 4.63 Sucrose 21.74 31.93 8.2615.33 19.35 82.18 Galactopinitol A 0 4.95 5.54 8.05 5.79 6.14Galactopinitol B 0 1.22 1.45 0 0 2.18 Fagopyritol B1 3.43 3.27 1.69 5.412.54 3.44 Galactinol 0 0 1.50 0 0 1.66 Raffinose 3.50 5.06 1.08 0 0 8.44Ciceritol 0 0 0 0 0 0 Fagopyritol B2 0 0 0 0 0 0 Stachyose 0 0 0 0 016.46

TABLE 15 Accumulation of soluble carbohydrates (μg/cm²) in soybeanleaves (1 cm² leaf disks) at 24 hours after feeding 50 mM myo-inositol,D-chiro-inositol, or D-pinitol, each in 1% sucrose solution, or 1%sucrose solution alone to stems of soybean explants 24 hours afterfeeding 50 mM cyclitol in 1% sucrose solution to explants D-chiro-Soluble myo-Inositol Inositol D-Pinitol Sucrose only Carbohydrate μg/cm²leaf area Fructose 88.82 121.03 62.53 24.85 Glucose 50.75 115.12 69.8221.14 D-Pinitol 147.24 124.80 757.97 133.76 D-chiro- 18.98 439.69 23.2912.21 Inositol myo-Inositol 296.44 10.15 5.60 25.31 Sucrose 46.10 29.5128.89 29.90 Maltose 9.34 9.85 4.11 11.59 Galactopinitol A 0 0 0 0Galactopinitol B 0 0 0 0 Fagopyritol 0 0 0 0 B1 Galactinol 0 0 0 0Raffinose 0 0 0 0 Ciceritol 0 0 0 0 Fagopyritol 0 0 0 0 B2 Stachyose 0 00 0

Some results and conclusions drawn from this series of experiments areas follows. Feeding myo-inositol, D-chiro-inositol, or D-pinitol tosoybean explants increased free myo-inositol 10 fold, freeD-chiro-inositol 35 fold, or D-pinitol 5 fold, respectively, in leaftissues at 24 hours after the start of feeding explants demonstratingthe uptake of cyclitols through the stem to the leaf via thetranspiration stream. Free D-chiro-inositol in leaf tissues wasincreased slightly after feeding myo-inositol or D-pinitol, but therewas no detection of galactosyl cyclitols, raffinose, or stachyose inleaf tissues indicating the absence of accumulation of these compoundsin leaves.

Feeding myo-inositol, D-chiro-inositol, or D-pinitol to soybean explantsincreased free myo-inositol 2 fold, free D-chiro-inositol 20 to 40 fold,or D-pinitol 2 to 4 fold, respectively, in seed coat tissues of dryseeds (14 days slow drying) demonstrating the movement of cyclitols tothe seed coat, presumably via the phloem. Feeding myo-inositol increasedD-chiro-inositol 5 to 10 fold and doubled raffinose and stachyose, withno increase in D-pinitol or galactopinitols in the seed coat, suggestingthat myo-inositol may directly serve as precursor for biosynthesis ofD-chiro-inositol or through D-pinitol as intermediate. FeedingD-chiro-inositol also increased fagopyritol B1 5 to 15 fold in seedcoats, but not other cyclitols, galactosyl cyclitols, or raffinose andstachyose. Feeding D-pinitol doubled galactopinitols and increasedD-chiro-inositol, fagopyritol B1, raffinose, and stachyose indicatingthat D-pinitol may serve as precursor to D-chiro-inositol biosynthesisand that galactopinitols may serve as a galactosyl donor for thebiosynthesis of stachyose.

Feeding myo-inositol, D-chiro-inositol, or D-pinitol to soybean explantsincreased free myo-inositol slightly, free D-chiro-inositol 15 to 40fold, or D-pinitol 4 to 15 fold, respectively, in axis tissues of dryseeds (14 days slow drying) demonstrating the downloading of cyclitolsfrom the seed coat to the embryonic axis. Feeding myo-inositol hadlittle effect on the accumulation of other soluble carbohydrates in theembryonic axis. Feeding D-chiro-inositol also increased fagopyritol B110 fold in seed coats, but not other cyclitols, galactosyl cyclitols, orraffinose and stachyose. Feeding D-pinitol doubled galactopinitols inthe embryonic axis, but not other cyclitols, galactosyl cyclitols, orraffinose and stachyose. These results suggest that galactopinitols andfagopyritols are biosynthesized by different pathways.

Feeding myo-inositol, D-chiro-inositol, or D-pinitol to soybean explantsdid not increase free myo-inositol, but increased free D-chiro-inositol5 to 6 fold, or D-pinitol 2 fold, respectively, in cotyledon tissues ofdry seeds (14 days slow drying) demonstrating the downloading ofcyclitols from the seed coat to the soybean embryo. Feeding myo-inositoldoubled free D-chiro-inositol but had little effect (or decreased) othersoluble carbohydrates consistent with myo-inositol being a precursor forthe biosynthesis of D-chiro-inositol. Feeding D-chiro-inositol alsoincreased fagopyritol B1 6 to 10 fold in cotyledons, but not othercyclitols, galactosyl cyclitols, or raffinose and stachyose, indicatingthat fagopyritols do not serve as galactosyl donors for stachyosebiosynthesis. Feeding D-pinitol did not increase accumulation ofcyclitols (other than D-pinitol), galactosyl cyclitols, raffinose, orstachyose in cotyledons.

These results are in general agreement with the those of Examples 2 and3.

Example 6 Buckwheat Plant Temperature Experiments

Common buckwheat (Fagopyrum esculentum Moench) belongs to Polygonoceaefamily. Originating in northeast Asia, southern Siberia and northernChina, there are 18 recognized natural species in Fagopyrum. Among them,common buckwheat is most important from economical, agricultural, andnutritional points of view. In buckwheat, the triangular fruit (achene)forms a single seed. The buckwheat embryo is rich in lipids (Horbowiczet al., J. Agric. Food Chem. 40:745-750 (1992), which is herebyincorporated by reference in its entirety), and high quality proteins(Elpidina et al., J. Exp. Bot. 41:969-977 (1990), which is herebyincorporated by reference in its entirety), and is embedded in a starchyendosperm (Marshall et al., Adv. Cereal Sci. Tech. 5:157-210 (1982);Steadman et al., J. Cereal Sci. 33:271-278 (2001), which are herebyincorporated by reference in their entirety).

Common buckwheat plants are dimorphic and heterostylous. One-half of theplants have pin-type flowers with long styles and short stamens, andone-half of the plants have thrum-type flowers with short styles andlong stamens (Marshall et al., Adv. Cereal Sci. Tech. 5:157-210 (1982),which is hereby incorporated by reference in its entirety). Each type isself-incompatible and cross-incompatible among plants with the sameflower type. Seed set requires legitimate cross pollination, pin bythrum and thrum by pin, by insects under field conditions or by handpollination in the greenhouse as in the present study (Horbowicz et al.,J. Agric. Food Chem. 40:745-750 (1992), which is hereby incorporated byreference in its entirety).

Buckwheat plants grow best in cool, moist climates. Daytime airtemperatures of 17° C. to 19° C. are optimal during flowering and seedmaturation of this plant (Marshall et al., Adv. Cereal Sci. Tech.5:157-210 (1982), which is hereby incorporated by reference in itsentirety). Because the crop matures in 10 to 12 weeks, it can be grownin temperate regions and higher altitude areas. The crop is sensitive tohigh temperatures and dry weather, especially when the plants areflowering (Slawinska et al., Seed Sci. Res. 11:223-233 (2001); Taylor etal., Crop Sci. 41:1792-1799 (2001), which are hereby incorporated byreference in their entirety).

Recent evidence points to the importance of special types ofcarbohydrates in development of seed desiccation tolerance andstorability (Koster et al., Plant Physiol. 88:829-832 (1998); Blackmanet al., Plant Physiol. 100:225-230 (1992); Horbowicz et al., Seed Sci.Res. 4:385-405 (1994); Obendorf et al., Seed Sci. Res. 7:63-74 (1997);Obendorf et al., Crop Sci. 38:78-84 (1998), which are herebyincorporated by reference in their entirety). During development oflegume seeds mainly sucrose and α-galactosides of sucrose areaccumulated (Horbowicz et al., Seed Sci. Res. 4:385-405 (1994); Obendorfet al., Seed Sci. Res. 7:63-74 (1997); Brenac et al., J. Plant Physiol.150:481-488 (1997), which are hereby incorporated by reference in theirentirety). Instead, buckwheat seeds contains sucrose and α-galactosidesof D-chiro-inositol (Horbowicz et al., Planta 205:1-11 (1998), which ishereby incorporated by reference in its entirety).

Six fagopyritols (galactosyl cyclitols), representing two distinctseries differing in bonding positions, were found in buckwheat seeds(Horbowicz et al., Planta 205:1-11 (1998); Steadman et al., J. CerealSci. 33:271-278 (2001); Steadman et al., Carbohydr. Res. 331:19-25(2001); Szczecinski et al., Bull. Pol. Acad. Sci. 46:9-13 (1998), whichare hereby incorporated by reference in their entirety). Fagopyritol B1and fagopyritol A1 are the major galactosides accumulated, andcorrelated to desiccation tolerance in buckwheat seeds (Horbowicz etal., Planta 205:1-11 (1998); Obendorf et al., Carbohydr. Res.328:623-627 (2000), which are hereby incorporated by reference in theirentirety). Structures of di- and trigalactosides of D-chiro-inositolhave been confirmed as well (Steadman et al., Carbohydr. Res. 331:19-25(2001), which is hereby incorporated by reference in its entirety). Allfagopyritols accumulate mainly in the embryo of buckwheat seeds, andmuch lower amounts in endosperm (Horbowicz et al., Planta 205:1-11(1998), which is hereby incorporated by reference in its entirety).

chiro-inositol plays a role in the biosynthesis ofgalactosamine-D-chiro-inositol, an insulin mediator in type II diabetes(Lamer et al., Biochem. Biophys. Res. Commun. 151:1416-1426 (1988);Romero et al., Adv. Pharmacology 24:21-50 (1993), which are herebyincorporated by reference in their entirety). In Type II (non-insulindependent diabetes mellitus) diabetic patients have deficiency of aninsulin mediator containing galactosamine-D-chiro-inositol phosphate(Asplin et al., Proc. Nat. Acad. Sci. 90:5924-5928 (1993), which ishereby incorporated by reference in its entirety). AddingD-chiro-inositol as a dietary supplement appeared to be effective inlowering symptoms of diabetes (Ortmeyer et al., Endocrinology132:640-645 (1993), which is hereby incorporated by reference in itsentirety). Several research groups are developing sources for naturaland synthetic supplies of D-chiro-inositol (U.S. Pat. No. 5,091,596 toKennigton et al; Mandel et al., J. Org. Chem. 58:2331-2333 (1993), whichare hereby incorporated by reference in their entirety). One naturalsource of D-chiro-inositol (in free form and as galactosides) isbuckwheat seed, and the bran milling fraction from buckwheat seed can beused for isolation and production of fagopyritols and freeD-chiro-inositol preparations for medical purposes (Obendorf et al.,Carbohydr. Res. 328:623-627 (2000); Steadman et al., J. Agric. FoodChem. 48:2843-2847 (2000); Horbowicz et al., J. Agric. Food Chem.40:745-750 (1992), which are hereby incorporated by reference in theirentirety).

Temperature during development of legume seeds had only minor effects onsoluble carbohydrate biosynthesis and accumulation (Gorecki et al., CropSci. 36:1277-1282 (1996); Obendorf et al., Crop Sci. 38:78-84 (1998),which are hereby incorporated by reference in their entirety). Howeverduring our preliminary studies, temperature during seed maturationaffected soluble carbohydrate content and composition of buckwheatembryos (Horbowicz et al., Planta 205:1-11 (1998), which is herebyincorporated by reference in its entirety). Warm temperature (25° C.)favored biosynthesis of sucrose, and embryos matured at cool temperature(18° C.) accumulated higher quantities of fagopyritol A1 and fagopyritolB1. During maturation of soybean embryos, warm temperature (25° C.)favors biosynthesis of fagopyritol B1, as well as sucrose, raffinose,D-chiro-inositol and D-pinitol (Obendorf et al., Crop Sci. 38:78-84(1998), which is hereby incorporated by reference in its entirety). Theobjective of this Example was to determine if temperature (15, 22 and30° C.) during buckwheat seed maturation in plants affects accumulationof soluble carbohydrates, dry and fresh mass, and germination ofbuckwheat embryos and seeds.

Materials and Methods

Buckwheat plants (cv. Mancan) were grown in the greenhouse at 24° C. day(14 hours) and 18° C. night (10 hours). Natural sunlight wassupplemented 14 hours daily with 740 μmol m² s⁻¹ light from 1000 WSylvania metal halide lamps. After opening first flowers, plants wereseparated into pin and thrum types and placed in separate growthchambers at 18° C. All plants received 14 hours of fluorescent lightdaily at about 300 μmol m² s⁻¹. After 7 to 10 days, plants were handpollinated by legitimate cross-pollination, pin×thrum and thrum×pin.Eight days after pollination the temperature in three growth chamberswas changed from 18° C. to 15° C., 22° C., and 30° C., respectively.Seeds were harvested at 8, 12, 16, 20, and 28 days after pollination(DAP) and analyzed for soluble carbohydrates. After the last harvest (28DAP) seeds were placed in a desiccator over saturated LiCl solution(RH=12%), and dried for 14 days before analysis. Weight of each groatwas measured. After drying over LiCl, seeds (four replications of 10groats each) were germinated on wet germination papers at 25° C. indarkness. After 2, 4, and 6 days the germination rate (in %) wasmeasured, as well as hypocotyl length.

Carbohydrates in buckwheat embryo were analyzed by high resolution gaschromatography as previously described (Horbowicz et al., Seed Sci. Res.4:385-405 (1994); Horbowicz et al., Planta 205:1-11 (1998), which areincorporated herein by reference in their entirety). Carbohydratestandards (sucrose, myo-inositol, fructose, glucose, raffinose andstachyose), internal standard (phenyl α-D-glucoside), pyridine andtrimethylsilylimidazole (TMSI) were purchased from Sigma. Fagopyritolstandards were purified from buckwheat (Horbowicz et al., Planta205:1-11 (1998); Steadman et al., Carbohydr. Res. 331:19-25 (2001),which are incorporated herein by reference in their entirety).Galactinol and D-chiro-inositol standards were a gift.

Results

Buckwheat embryos accumulated maximum fresh weight by 20 days afterpollination (DAP) when matured at 15° C., by 16 DAP when matured at 22°C., and by 12 DAP when matured at 30° C. (Table 16).

TABLE 16 Dry weight (DW) and fresh weight (FW) of buckwheat embryos(mg/embryo) from seeds matured at 15, 22, or 30° C. as a function ofdays after pollination (DAP). Values are mean ± SE for three replicatesamples. Maturation Maturation Maturation at 15° C. at 22° C. at 30° C.FW DW FW DW FW DW DAP (mg) (mg) (mg) (mg) (mg) (mg) 8 0.99 ± 0.24 ± 0.99± 0.24 ± 0.99 ± 0.24 ± 0.08 0.12 0.08 0.07 0.08 0.07 12 3.00 ± 0.70 ±6.77 ± 1.47 ± 11.17 ± 4.43 ± 0.71 0.27 1.41 0.14 0.95 0.43 16 11.50 ±4.23 ± 14.13 ± 5.77 ± 10.57 ± 5.17 ± 1.25 1.07 3.06 1.62 0.20 0.23 2017.37 ± 8.67 ± 13.43 ± 6.97 ± 9.90 ± 7.63 ± 0.64 0.32 0.67 0.61 0.520.26 28 14.43 ± 8.03 ± 8.60 ± 6.37 ± 6.67 ± 6.83 ± 1.07 0.26 0.38 0.130.87 0.65 28 DAP + 7.80 ± 7.07 ± 7.40 ± 6.50 ± 9.73 ± 6.93 ± 2 wk 12%0.72 0.64 0.98 0.86 0.59 0.54 RHHighest daily increase in fresh weight occurred between 12 and 16 DAPwhen matured at 15 and 22° C. and between 8 and 12 DAP when matured at30° C.

Independently of maturation temperature, the dry weight of embryosreached maximal values after 20 DAP, but fastest daily increase of DWoccurred between 8 and 12 DAP at 30° C., between 12 and 16 DAP at 22°C., and at 15° C. between 16 and 20 DAP (Table 16). Although differencesin the rates of dry matter accumulation occurred between alltemperatures, the final dry weight of embryos matured at 15, 22 and 30°C. was similar. The slight decrease of dry weight in embryos matured at15° C. noted after 2 weeks of drying over LiCl solution probably was theeffect of difficulty in removing all remnants of cotyledons surroundedby endosperm tissue. Equal accumulation of embryo dry weight was alsonoted in our previous experiments, where seeds were matured in 18 and25° C. (Horbowicz et al., Planta 205:1-11 (1998), which is herebyincorporated by reference).

Mean dry weight of groats gradually declined when maturation temperatureincreased. Mean dry weight of buckwheat groats matured at 15° C. was48.17±1.75 mg, at 22° C.-41.27±1.48 mg, and at 30° C.-35.20±1.31 mg.Data presented here are the groat mean (±SE) dry weights from 50 seeds.Calculated average decline of buckwheat groat weight with increasingtemperature was −0.86 mg/1° C.

Maturation temperature had no effect on the total amount of solublecarbohydrates in buckwheat embryos (Table 17).

TABLE 17 Soluble carbohydrates (μg/embryo) in buckwheat embryos fromseeds matured at 15, 22, or 30° C. All seed harvested at 28 days afterpollination (DAP) and dried 2 weeks at 12% RH. Values are mean ± SE forthree replicate samples. Sol. Maturation Maturation Maturationcarbohydrate at 15° C. at 22° C. at 30° C. D-chiro- 9.76 ± 2.86 6.81 ±1.07 3.49 ± 0.66 Inositol Fagopyritol A1 45.59 ± 5.24  34.15 ± 10.0221.78 ± 1.55  Fagopyritol B1 256.00 ± 38.30  219.15 ± 24.20  159.60 ±7.70  Fagopyritol A2 3.66 ± 0.84 11.12 ± 3.04  15.52 ± 0.68  FagopyritolB2 2.47 ± 0.74 12.98 ± 3.59  19.70 ± 1.74  Sub total 317.50 ± 48.00 284.60 ± 42.00  220.10 ± 12.30  myo-Inositol 2.91 ± 0.54 5.03 ± 0.743.25 ± 0.64 Galactinol 0 1.57 ± 0.79 1.60 ± 0.04 Digalactosyl 0.25 ±0.25 0.55 ± 0.55 1.69 ± 0.73 myo-inositol Sub total 3.16 ± 0.79 7.15 ±2.08 6.54 ± 1.41 Sucrose 225.00 ± 14.90  250.20 ± 22.70  376.40 ± 59.20 Total soluble 545.70 ± 63.70  542.00 ± 66.80  603.00 ± 72.90 carbohydratesReducing sugars, fructose and glucose were present only in early stagesof embryo development (8 and 12 DAP). Sucrose slightly decreased between8 and 12 DAP, probably due to temperature and pollination shocks, andthen during next 4 days increased dramatically reached maximal values 16DAP (FIG. 23A). This increase was due to a rapid increase of embryofresh weight during maturation at 15 and 22° C., but not at 30° C. (FIG.23A and Table 16). During maturation at 30° C., the highest dailyincrease of fresh weight occurred between 8 and 12 DAP, but in the sametime sucrose level slightly declined. After 16 DAP sucrose level inembryos matured at 15 and 22° C. decreased, and finally after dryingover LiCl solution, the embryo sucrose content was 225.0 and 250.2μg/embryo, respectively. Maturation at 30° C. and further drying overLiCl solution of buckwheat embryos did not change the level of sucrose,which remained much higher at 376.4 μg (Table 17).

Monogalactosides of D-chiro-inositol (isomers fagopyritol A1 andfagopyritol B1) were the dominant soluble carbohydrates in embryos ofbuckwheat seeds matured in 15° C., but not when matured at 22 or 30° C.(FIGS. 23B and C). After drying of harvested buckwheat seeds at 12%relative humidity (RH) over LiCl solution, the ratio of fagopyritol B1to sucrose was 1.14:1 when embryos were matured at 15° C., 0.88:1 inembryos matured at 22° C., and only 0.43:1 in embryos matured at 30° C.(Table 17). A similar situation, a clear decline of sucrose in relationto increased temperature, occurred in the case of positional isomerfagopyritol A1, although level of fagopyritol B1 was 5 to 7 times higherthan fagopyritol A1 (FIGS. 23B and C and Table 17).

An opposite situation occurred in the case of D-chiro-inositoldigalactosides, fagopyritol A2 and fagopyritol B2 (FIGS. 24A-C); higheramounts accumulated in embryos matured at higher temperatures (22 and30° C.) than at 15° C. After 2 weeks of drying of buckwheat seeds,embryos of seeds matured at 30° C. contained about 4 times morefagopyritol A2, and almost 8 times more fagopyritol B2 than embryos ofseeds matured at 15° C. (Table 17). Similar effect of maturationtemperature was found in case galactosides of myo-inositol (FIGS.25A-C). Accumulation of myo-inositol in embryo was similar at alltemperatures of buckwheat seed maturation, however the amount of itsgalactosides (galactinol and digalactosyl myo-inositol (DGMI)) was muchless in embryos of seeds matured at 15° C. than in embryos of seedsmatured at 22° C. and especially in seeds matured at 30° C. (Table 18).

TABLE 18 Minor soluble carbohydrates (μg/embryo) in buckwheat embryosfrom seeds matured at 15, 22, or 30° C. as a function of days afterpollination (DAP). Values are mean ± SE for three replicate samples.Soluble Maturation 28 DAP + 2 wk Carbohydrate temperature 16 DAP 20 DAP28 DAP 12% RH Digalactosyl 15° C. 0 0 0 0.25 ± 0.25 myo-inositol 22° C.0 1.38 ± 0.84 1.12 ± 0.18 0.55 ± 0.55 30° C. 1.02 ± 1.02 3.54 ± 2.512.21 ± 0.32 1.69 ± 0.73 Fagopyritol A3 15° C. 0 0 0 0 22° C. 0 0 30° C.9.80 ± 5.47 4.41 ± 4.41 Raffinose 15° C. 0 0 0 0 22° C. 0 0.78 ± 0.4530° C. 0.70 ± 0.12 1.21 ± 0.95 Stachyose 15° C. 0 0 0 0 22° C. 0 3.04 ±3.04 30° C. 2.71 ± 2.71 5.07 ± 2.53

During later stages of buckwheat embryo development (after 20 DAP and 28DAP) at 22 and 30° C., small amounts of raffinose and stachyose werefound (Table 18). In embryos matured in 30° C., fagopyritol A3 (atrigalactoside of D-chiro-inositol) was present as well. Embryos maturedin 15° C. did not contain these carbohydrates in measurable quantities(Table 18). After 2 weeks dehydration of buckwheat seeds, analyzedembryo raffinose, stachyose, and fagopyritol A3 declined to levels belowthe limit of detection.

The germination rate of seeds matured in low temperatures (15 or 22° C.)was lower than for seeds matured at 30° C. (FIG. 26A). Differences werequite clear after 4 and 6 days of germination on moist germination paperin darkness and 25° C. Germination rate of seeds matured in 22° C. was14, 18, and 20% lower after 2, 4, and 6 days respectively, than forseeds matured at 15° C. When compared to seeds matured at 30° C., thegermination rate of seeds matured in 22° C. was 20%, 44%, and 41% lower.Germination rate of seeds matured in 30° C. was similar to those maturedin 15° C. after 2 days of germination, however after 4 and 6 days, seedsmatured at 30° C. germinated 90%, and seeds matured at 15° C. germinatedonly 66% and 71% (FIG. 26A).

Growth of hypocotyls in germinating buckwheat seeds was faster in seedsmatured at 15 and 22° C., than for seeds matured at 30° C. (FIG. 26B).Such a situation occurred after 2 and 4 days of germination process, butafter 6 days the differences in hypocotyl length were not significant.

Discussion

The response of plants to stress involves complex physiological andbiochemical responses. Conditions during seed development and maturationcan have an impact on subsequent seed quality. Soil moisture andtemperature stress in that time has been suggested to have an influenceon seed and seedling vigor. Factors during seed maturation such asenvironmental conditions also have an impact on seed viability (Baskinet al., Seeds: Ecology, Biogeography, and Evolution of Dormancy andGermination, Academic Press, New York, pp. 41-43 (1998), which is herebyincorporated by reference in its entirety). High temperatures duringgrowth can increase biochemical reactions in plants, but it might notalways be transferred to higher productivity because of heat stressconstraints such as limited water supply, increase in leaf temperature,increased respiration, decline of the synthesis and/or activity ofphotosynthetic enzymes. In buckwheat groats matured in high temperatures(22 or 30° C.), reduced mean weight was noted, than when produced in lowtemperature (15° C.). Although high temperature maturation (30° C.) canchange physiological reactions the buckwheat embryos obtained in suchconditions have similar dry weight to those from matured in lowertemperatures (15 or 22° C.). Dry weight of whole seed was lower, mainlydue to decrease of endosperm deposition (Horbowicz et al., Planta205:1-11 (1998), which is hereby incorporated by reference in itsentirety). Additionally, plants growing at 25° C. produced only half asmany seeds as plants at 18° C. (Slawinska et al., Seed Sci. Res.11:223-233 (2001), which is hereby incorporated by reference in itsentirety). All mentioned facts can have huge impact on buckwheat seedyield. Probably, the difference in temperature during buckwheatflowering and seed filling is the main factor influencing the largevariability in seed set and seed yield among years (Slawinska et al.,Seed Sci. Res. 11:223-233 (2001); Taylor et al., Crop Sci. 41:1792-1799(2001), which is hereby incorporated by reference in its entirety).

During high temperature stressed plants make a special proteins calledheat shock proteins (HSPs). Among the different HSPs produced by plants,the small (sm) HSPs appear to be particularly important because of theirabundance. In addition, smHSPs are expressed during specific stages ofplant development including seed maturation, indicating they alsofunction in the absence of stress to protect components essential forseed development (Schoffl et al., Plant Physiol. 117:1135-1141 (1998),which is hereby incorporated by reference in its entirety). HSPs showinga reversible interaction with other proteins and preventing eithercomplete denaturation or supporting proper folding of enzymes under orafter protein denaturing conditions. Some HSP-like proteins are involvedin the processes of targeting other proteins to organelles or to theirsuborganellar localization and a number of HSPs are expressed in theabsence of external stressors, during embryogenesis and seed maturationin many plant species (Schoffl et al., Acta Physiol. Plantarum19:549-556 (1997), which is hereby incorporated by reference in itsentirety).

It is possible that HSPs might have an influence on biosynthesis ofcarbohydrates during maturation of buckwheat embryos. In buckwheatembryos matured in higher temperatures biosynthesis of fagopyritols B1and its positional isomer fagopyritol A1 was partly inhibited (Horbowiczet al., Planta 205:1-11 (1998), which is hereby incorporated byreference in its entirety). In present studies total amounts of bothfagopyritols in embryos matured at 15° C. is about twice as high asthose matured at 30° C. However, sucrose level is much higher inbuckwheat embryos matured at high temperatures. This observation differsfrom soybean embryos, where maturation at 25° C. enhanced the amount offagopyritol B1 when compared to embryos matured at 18° C. (Obendorf etal., Crop Sci. 38:78-84 (1998), which is hereby incorporated byreference in its entirety).

D-chiro-inositol and its galactosides (fagopyritols) have potentialmedical importance in lowering symptoms of non-insulin dependentdiabetes mellitus (Asplin et al., PNAS USA 90:5924-5928 (1993); Lamer etal., Biochem. Biophys. Res. Commun. 151:1416-1426 (1988); Ortmeyer etal., Endocrinology 132:640-645 (1993); Romero et al., Adv. Pharmacology24:21-50 (1993), which are hereby incorporated by reference in theirentirety). Buckwheat flour produced from seeds matured at lowtemperature (15 or 18° C.) is therefore more valuable than from seedsmatured at 22 or 30° C. Buckwheat seeds can be an excellent and naturalsource for production of medicines used by diabetes patients (U.S. Pat.No. 6,162,795 to Obendorf et al; U.S. Pat. No. 6,492,341 to Obendorf etal., which are hereby incorporated by reference in their entirety).

High temperature during buckwheat seed maturation enhanced thebiosynthesis of di-α-galactosides of D-chiro-inositol (fagopyritol A2and fagopyritol B2) and α-galactosides of sucrose (raffinose andstachyose). This observation is opposite to our earlier results, whereincreased level of sucrose galactosides was noted in buckwheat embryosof seeds matured at 18° C. in comparison to embryos from seeds maturedat 25° C. (Horbowicz at al., Planta 205:1-11 (1998), which is herebyincorporated by reference in its entirety). Similarly, in the presentstudy, a higher level of galactinol, the substrate for biosynthesis ofraffinose and stachyose, was found in buckwheat embryos matured athigher temperatures. Galactinol is the galactosyl donor for bothraffinose and stachyose biosynthesis, as well as the digalactoside ofmyo-inositol. According to Castillo et al., J. Agric. Food Chem.38:351-355 (1990), which is hereby incorporated by reference in itsentirety, low temperature during soybean seed maturation promotesgalactinol biosynthesis. In buckwheat is the opposite situation—hightemperature promotes accumulation of galactinol, raffinose, andstachyose. Based on that it was concluded that physiological response totemperature stress during seed maturation in buckwheat is different thanwhat occurs in legumes (Castillo et al., J. Agric. Food Chem. 38:351-355(1990); Gorecki et al., Crop Sci. 36:1277-1282 (1996), which are herebyincorporated by reference in their entirety). In fact, for growing oflegumes, high temperatures are needed, whereas for buckwheat, dailytemperatures 17 to 19° C. are optimal.

Surprisingly, germination was higher in case of buckwheat seeds maturedat 30° C. than for those matured at 15 or 22° C. Lowest germination ratewas found in seeds matured at 22° C. Possibly during maturation ofbuckwheat seeds at 22° C. germination inhibitors are biosynthesized inhigher concentration and they affect the proteolytic enzymes duringgermination (Belozersky et al., J. Plant Physiol. 46(3):330-339 (1999),which is hereby incorporated by reference in its entirety). Seedsmatured at 15° C. have delayed maturation, and therefore inhibitors areprobably absent or in low, insufficient quantities. At 30° C. seedsmature very fast and it is quite possible that these seeds have lowerlevels of germination inhibitors, due to the shorter time of maturation.

Example 7 Buckwheat Explant Feeding Experiments

Buckwheat explants, consisting of a stem segment with attached leaf andterminal floral cluster, were patterned after the soybean explantsdescribed in Example 4. This example uses the buckwheat explant systemto study the transport of cyclitols, fed through the stem, to thedeveloping buckwheat seed and their incorporation into fagopyritols.D-chiro-inositol, D-pinitol, or myo-inositol (100 mM in 1% sucrose) or1% sucrose (without cyclitols) were fed to buckwheat explants throughthe stem for 5 days and then the seeds were slow dried. Solublecarbohydrates were extracted and analyzed from embryos of the seeds andfrom leaf disks. The results are shown in Tables 19-25, below.

TABLE 19 Soluble carbohydrates (μg/embryo) in embryos of seeds frombuckwheat explants fed 100 mM D-chiro-inositol in 1% sucrose solution -feeding 5 days before slow drying (micrograms/embryo) After 2 days 4days 7 days Soluble After 1 day 5 days slow slow slow Carbohydratefeeding feeding drying drying drying D-Pinitol 0 0 0 0 0 0 0 0 0 0D-chiro-Inositol 2.28 93.10 1.32 2.20 56.97 14.89 87.78 25.03 44.5541.29 122.37 138.86 55.10 myo-Inositol 1.63 2.03 1.84 3.29 2.20 1.714.59 1.88 2.64 1.20 2.74 2.19 1.10 Sucrose 66.64 268.14 124.83 202.99162.68 232.10 90.04 185.14 147.00 121.44 202.20 118.11 121.99Galactopinitol A 0 0 0 0 0 0 0 0 0 0 0 0 0 Galactopinitol B 0 0 0 0 0 00 0 0 0 0 0 0 Fagopyritol A1 0 10.71 25.53 27.38 41.11 0 133.47 117.3242.86 21.71 69.49 52.16 27.96 Fagopyritol B1 0 33.64 127.23 125.16298.43 0 623.00 635.89 388.66 190.64 322.50 270.67 208.33 Galactinol 03.15 9.84 6.82 7.21 0 3.75 3.63 3.90 1.71 2.62 2.70 0 Fagopyritol A2 0 06.43 4.19 2.57 0 10.11 4.91 2.04 1.19 1.93 4.42 1.33 Fagopyritol B2 0 05.51 3.95 2.28 0 5.48 3.45 2.83 0.91 1.72 1.57 1.99 Digalactosyl 0 0 0 00 myo-inositol 0 0 0 0 0 0 0 0

TABLE 20 Soluble carbohydrates (μg/embryo) in embryos of seeds frombuckwheat explants fed 100 mM D-pinitol in 1% sucrose solution - feeding5 days before slow drying (micrograms/embryo) After 2 days 4 days 7 daysSoluble After 1 day 5 days slow slow slow Carbohydrate feeding feedingdrying drying drying D-Pinitol 35.53 204.10 230.83 134.07 148.04 263.02163.47 162.97 85.34 328.27 56.90 173.51 129.52 D-chiro-Inositol 4.501.32 1.51 2.19 1.97 28.25 0.70 4.78 1.01 9.41 0.94 1.11 7.71myo-Inositol 1.00 1.38 6.63 5.34 2.78 4.60 3.07 4.92 3.38 3.43 1.37 4.813.33 Sucrose 212.67 89.73 106.43 87.98 84.50 86.41 167.61 229.72 122.05147.85 84.96 104.05 177.42 Galactopinitol A 0 0 0 3.39 6.41 6.18 12.91 09.09 5.84 6.68 0 0 Galactopinitol B 0 1.61 4.39 5.88 0 1.10 0 0 1.811.90 0 0 0 Fagopyritol A1 0 0 2.38 16.78 10.82 21.43 29.81 39.10 12.4422.91 23.96 15.80 20.01 Fagopyritol B1 1.44 0.98 7.29 116.5 64.64 90.76165.96 165.56 77.20 75.14 111.41 77.90 119.44 Galactinol 2.51 0 10.692.68 0 3.26 7.75 4.14 1.24 3.40 2.79 3.31 0 Fagopyritol A2 0 0 0 6.181.30 0 9.52 6.64 0.71 4.65 6.89 4.73 2.21 Fagopyritol B2 0 0 0 5.70 1.600 6.69 4.29 0.48 4.98 4.46 2.47 Digalactosyl 0 0 0 0 0 myo-inositol 0 01.05 0 0 0 0 0

TABLE 21 Soluble carbohydrates (μg/embryo) in embryos of seeds frombuckwheat explants fed 100 mM myo-inositol in 1% sucrose solution -feeding 5 days before slow drying (micrograms/embryo) After 2 days 4days 7 days Soluble After 1 day 5 days slow slow slow Carbohydratefeeding feeding drying drying drying D-Pinitol 0 0 0 tr tr tr 0 tr trD-chiro-inositol 6.45 15.19 6.42 3.54 14.01 126.48 1.07 6.67 9.90myo-Inositol 4.56 4.93 1.67 3.66 2.37 4.72 0 2.27 2.11 Sucrose 306.16225.64 313.35 180.43 163.04 74.48 66.92 161.20 121.70 Galactopinitol A 00 0 0 0 0 0 0 0 Galactopinitol B 0 0 0 0 0 0 0 0 0 Fagopyritol A1 018.90 62.19 19.72 14.49 10.94 29.81 41.42 34.61 Fagopyritol B1 0 95.33300.84 111.78 79.17 44.47 129.87 183.36 188.73 Galactinol 0 19.19 10.368.41 2.09 3.71 3.46 4.79 0 Fagopyritol A2 0 2.60 38.72 11.49 2.54 010.89 4.52 2.64 Fagopyritol B2 0 1.62 35.81 13.37 3.53 0 3.22 3.31 2.57Digalactosyl 0 0.85 6.63 2.13 0.35 myo-inositol 0 8.61 0 0

TABLE 22 Soluble carbohydrates (μg/embryo) in embryos of seeds frombuckwheat explants fed 1% sucrose (without cyclitols) solution - feeding5 days before slow drying (micrograms/embryo) After 2 days 4 days 7 daysSoluble After 1 day 5 days slow slow slow Carbohydrate feeding feedingdrying drying drying D-Pinitol 0 0 0 0 tr tr 0 tr tr D-chiro-Inositol3.22 5.57 1.94 14.09 24.47 8.02 2.09 5.09 26.74 myo-Inositol 1.50 3.636.44 2.46 17.80 2.71 2.94 4.07 2.30 Sucrose 211.90 111.20 189.33 177.61524.20 141.36 105.55 246.49 151.69 Galactopinitol A 0 0 0 0 tr 0 0 0 trGalactopinitol B 0 0 0 0 tr 0 0 0 0 Fagopyritol A1 0.70 12.27 16.0223.01 27.67 51.20 14.64 15.97 76.65 Fagopyritol B1 0.87 55.08 89.92138.32 151.69 237.27 72.98 111.37 476.65 Galactinol 1.47 15.87 26.38 019.03 10.02 8.45 5.93 5.03 Fagopyritol A2 0 0.58 10.60 7.35 8.12 12.296.87 6.34 8.09 Fagopyritol B2 0 0.66 11.05 7.65 6.72 10.83 5.72 6.344.39 Digalactosyl 0 0. 2.98 0.82 myo-inositol 3.02 0.60 0.74 1.48 0

TABLE 23 Soluble carbohydrates (μg/10 mg leaf disk) in leaves frombuckwheat explants fed 100 mM D-chiro-inositol in 1% sucrose solution -leaf composition, micrograms in 10 mg disc Soluble After 1 hour After 24hours After 72 hours Carbohydrate feeding feeding feeding Fructose 2.0519.96 115.25 Glucose 3.90 16.53 77.78 D-Pinitol 0 0 0 D-chiro-inositol2.06 21.16 72.91 myo-Inositol 3.76 1.88 3.91 Sucrose 16.50 15.13 16.15

TABLE 24 Soluble carbohydrates (μg/10 mg leaf disk) in leaves frombuckwheat explants fed 100 mM D-pinitol in 1% sucrose solution - leafcomposition, micrograms in 10 mg disc Soluble After 1 hour After 24hours After 72 hours Carbohydrate feeding feeding feeding Fructose 53.8260.78 42.84 Glucose 35.78 48.92 35.53 D-Pinitol 2.45 121.45 64.73D-chiro-Inositol 3.42 4.82 3.71 myo-Inositol 4.46 3.05 4.23 Sucrose84.83 2.18 9.04

TABLE 25 Soluble carbohydrates (μg/10 mg leaf disk) in leaves frombuckwheat explants fed 1% sucrose (without cyclitols) solution - leafcomposition, micrograms in 10 mg disc Soluble After 1 hour After 24hours After 72 hours Carbohydrate feeding feeding feeding Fructose 4.0520.25 23.35 Glucose 4.63 10.35 8.01 D-Pinitol 0 0 0 D-chiro-Inositol3.51 3.81 6.26 myo-Inositol 4.21 5.58 8.45 Sucrose 39.23 14.29 17.40

Based on the above data it was determined that feeding D-chiro-inositolto buckwheat explants increased free D-chiro-inositol 40 fold in leavesdemonstrating the transport of cyclitols to leaves via the transpirationstream. Feeding D-pinitol to buckwheat explants increased free D-pinitoldramatically in leaves. D-Pinitol does not accumulate in buckwheatleaves or seeds of explants fed D-chiro-inositol, myo-inositol, orsucrose without cyclitols. Galactosyl cyclitols, raffinose, and sucrosedo not accumulate in leaf tissues. Feeding D-chiro-inositol to buckwheatexplants increased free D-chiro-inositol 3 to 10 fold and fagopyritol B12 fold in embryos of buckwheat seeds demonstrating the transport ofD-chiro-inositol to buckwheat seeds and its incorporation intofagopyritols. Feeding D-pinitol to buckwheat explants increased freeD-pinitol in buckwheat embryos demonstrating the transport of D-pinitolto seeds and embryos; these embryos did not accumulate galactopinitols,indicating that buckwheat does not have the enzymes for accumulation ofgalactopinitols. Signals corresponding to galactopinitol retention timeswere similar to background signals. Presence of galactopinitols couldnot be verified. If present, galactopinitols were present only in traceamounts. Results of these experiments further demonstrate thatfagopyritols and galactopinitols are biosynthesized by differentpathways.

Example 8 Biosynthesis of an Insulin Mediator

Growth of Recombinant E. coli and Isolation of Recombinant Proteins

cDNAs corresponding to the FeGolS-1, FeGolS-2, and GmGolS genes wereinserted into pET-14B expression vectors. The vector also contained agene for ampicillin resistance and a sequence that codes for sixhistidines on the N-terminal end of the expressed protein. The vectorscontaining the gene inserts were used to transform E. coli strain BL21,containing the bacteriophage lysogen DE3. The bacteria were thenstreaked on ampicillin-containing plates and incubated overnight (8-12hours) at 37° C. One colony from each plate was then transferred to 2 mLof Luria Broth (LB) containing 0.05 mM ampicillin in 10 mL screw-cappedPyrex tubes. The tubes were then placed in an incubator at 37° C. withshaking at 175 rpm overnight (8-12 hours). One mL of the startercultures was then transferred to 250 mL of the LB-Amp solution and grownunder the same conditions for three hours. After three hours, IPTG wasadded to induce expression of the genes in the pET-14B vector. Thebacteria were then grown for another three hours and harvested viacentrifugation at 6,000 rpm. Bacteria from 500 mL of LB-Amp were lysedusing 5 mL of BugBuster™ solution. Nucleic acids and non-solublecellular matter were removed from the crude extract by centrifugationand filtration and the soluble extract was then loaded onto a Ni²⁺-NTAcolumn. The target proteins with the N-terminal histidine tag bound tothe column while all other soluble proteins were washed away. Theseenzymes were eluted from the column by the addition of imidazolecontaining extraction buffer. The protein solution was dialyzed against5 mM Mn²⁺ solution and then used for enzyme assays.

Enzyme Assays

Assays were completed under varying conditions to begin to characterizethe purified galactinol synthase enzymes. Assays were first designed todetermine if the enzymes could synthesize galactinol and fagopyritols(A). The optimal concentration of Mn²⁺ for enzyme action was thendetermined (B). The enzymes were next used in assays to determine theirsubstrate specificity (C). Finally, assays were completed to determinethe reaction kinetics of the enzymes (D).

(A) Initial Assays of Purified Recombinant FeGolS-1, FeGolS-2, andGmGolS Enzymes:

It was first determined that the purified recombinant FeGolS-1,FeGolS-2, and GmGolS enzymes could synthesize fagopyritols andgalactinol. To determine galactinol synthase activity, assays werecompleted using myo-inositol as the galactosyl acceptor andUDP-galactose as the galactosyl donor. Approximately 1-2 μg of eachenzyme was added to a 50 μL solution containing 20 mM myo-inositol, 20mM UDP-galactose, 50 mM HEPES, pH 7.0, 2 mM DTT, and 3 mM Mn²⁺ (MnCl₂)at 30° C. The reactions were stopped after 3 hours with the addition of50 μL of 100% EtOH. To determine fagopyritol synthase activity, the samereaction conditions were used, except D-chiro-inositol was used as thegalactosyl acceptor instead of myo-inositol.

(B) Optimal Concentration of Mn²⁺:

To determine the concentration of Mn²⁺ in which the enzymes had thegreatest activity, multiple assays were completed varying the amount ofMn²⁺. Earlier studies of galactinol synthase enzymes from other plantsreported optimal Mn²⁺ concentrations ranging from 1 mM to 15 mM. Twodifferent sets of assays were completed, one using myo-inositol as thegalactosyl acceptor, and the other using D-chiro-inositol. In both sets,1-2 μg of each enzyme was added to a 50 μL solution containing 20 mMgalactosyl acceptor, 20 mM UDP-galactose, 50 mM HEPES, pH 7.0, 2 mM DTT,and varying Mn²⁺ concentrations of 0, 1, 3, 5, 10 and 15 mM, at 30° C.After 3 hours, the reactions were stopped with the addition of 50 μL of100% EtOH.

(C) Substrate Specificity Assays:

The substrate specificity of the three galactinol synthase enzymes wascharacterized through assays varying the galactosyl acceptor.myo-inositol, D-chiro-inositol, pinitol, L-chiro-inositol, ononitol,bornesitol, sequoyitol, quebrachitol, epi-inositol and scyllo-inositolwere used as substrates in reactions with all three enzymes. Thereactions were completed using 1-2 μg of enzyme in a 50 μL solutioncontaining 20 mM galactosyl acceptor, 20 mM UDP-galactose, 50 mM HEPES,pH 7.0, 2 mM DTT, 5 mM Mn²⁺ at 30° C. After 3 hours, the reactions werestopped with the addition of 50 of 100% EtOH.

(D) Reaction Kinetics:

The assays to determine the K_(m) and V_(max) of the enzymes in thesynthesis of galactinol from myo-inositol and UDP-galactose were set upas follows:

-   -   Reaction A: 5 mM myo-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂    -   Reaction B: 10 mM myo-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂    -   Reaction C: 15 mM myo-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂    -   Reaction D: 20 mM myo-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂    -   Reaction E: 25 mM myo-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂

The assays to determine the K_(m) and V_(max) of the enzymes in thesynthesis of fagopyritol A1 and fagopyritol B1 from D-chiro-inositol andUDP-galactose were set up as follows:

-   -   Reaction A: 5 mM D-chiro-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂    -   Reaction B: 10 mM D-chiro-inositol        -   20 mM UDP-Galactose        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂    -   Reaction C: 15 mM D-chiro-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂    -   Reaction D: 20 mM D-chiro-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂    -   Reaction E: 25 mM D-chiro-inositol        -   20 mM UDP-Galactose        -   1 mM DTT        -   50 mM Hepes, pH 7.0        -   5 mM MnCl₂

To each reaction, ˜4-5 μg of enzyme were added. Each reaction was runfor 0, 3, 6, 9, and 12 minutes at 30° C. The reactions were stopped withthe addition of 50 μL of 100% EtOH and 25 μL of internal standard. Thereactions were then filtered through Nanosep tubes and 100 μL of eachreaction added to silyation vials. Samples were dried under nitrogen andstored over P₂O₅ overnight. Dry residues were derivatized with 100 μL oftrimethylsilylsylimadazole:pyridine (1:1, v/v) at 80° C. for 45 minutes,and 1 μL was injected for GC analysis of products as previouslydescribed (Horbowicz et al., Planta 205:1-11 (1998), which is herebyincorporated by reference in its entirety) using an HP1-MS capillarycolumn.

All five reactions were plotted on a product concentration vs. timeplot. The concentration of the enzyme had to be small enough so that thereaction was still linear after six minutes. The V_(o) for each reactionwas determined by finding the slope of this linear portion of the curve(i.e. if its linear, use the zero point and the concentration of productafter three minutes to calculate the slope of that portion of thereaction). Once this was completed, V_(o) (Rate) versus myo-inositolconcentration was plotted. Finally, a Lineweaver-Burke Plot was made byplotting 1/V_(o) vs. 1/[substrate]. If the line was linear, then itsslope was the K_(m)/V_(max). The y-intercept was 1/V_(max), thex-intercept was −1/K_(m).

All samples from the assays were analyzed by gas chromatography. Allwere prepared for analysis in the same way. After addition of 50 μL of100% EtOH, 25 of Internal Standard (25 μg of phenyl α-D-glucoside) wasadded to the reaction mixture. The solution was then filtered usingNanoSep tubes and 100 μL was transferred to a silation vial. The sampleswere then dried under nitrogen and desiccated over P₂O₅ overnight. Thedried samples were then derivitized with 100 μL of TMSI:pyridine (1:1,v/v) and then analyzed by gas chromatography.

Synthesis of the Putative Insulin Mediator

In order to synthesize the putative insulin mediator, it is necessary tofirst synthesize UDP-galactosamine. Work was completed developing aprotocol for the synthesis of the compound and its purification for usein further assays. UDP-galactosamine was synthesized fromgalactosamine-1-phosphate (from Sigma). The synthesis was done using theprocedure outlined in Heidlas et al., J. Org. Chem. 57:152-157 (1992),which is hereby incorporated by reference in its entirety. The procedureuses an uridyltransferase (EC 2.7.7.9) to transfer a UDP moiety fromUDP-glucose to galactosamine-1-phosphate to make UDP-galactosamine on agram scale (FIG. 27). The UDP-galactosamine synthesized in the reactionwas purified and desalted using a Bio-Rad P-2 Gel column. The fractionscontaining UDP-galactosamine were analyzed by HPLC using an AlltechEconosil C18 10U column (250 mm length, 4.6 mm I.D.) and avariable-wavelength detector at 254 nm. The mobile buffer was 20 mM TEAA(triethyl ammonium acetate buffer, pH 7.0) with an increasing gradientof acetonitrile (0-4% acetonitrile) after 30 minutes to clean the column(Rabina et al., Glycoconjugate J. 18:799-805 (2001), which is herebyincorporated by reference in its entirety). Identification was basedupon retention times determined earlier with known substrates and thedeveloped separation method. Fractions containing UDP-galactosamine wereconcentrated by freeze drying, and the lyophilized powder containingUDP-galactosamine was resuspended in 1 mL of water. The purifiedUDP-galactosamine and D-chiro-inositol can now be used as substrates forthe recombinant FeGolS-2 enzyme to biosynthesize the insulin mediatorgalactosamine D-chiro-inositol (FIG. 28). Two products are expected:2-amino-2-deoxy-α-D-galactosamine-(1-3)-1D-chiro-inositol (a putativeinsulin mediator) and2-amino-2-deoxy-α-D-galactosamine-(1-2)-1D-chiro-inositol (isomer of theputative insulin mediator) in addition to UDP. Initial determination ofsuccessful synthesis can be assayed by gas chromatography. The peakscorresponding to fagopyritol A1, fagopyritol B1, D-chiro-inositol, andmany other soluble carbohydrates are known, and the two galactosamineD-chiro-inositol products should correspond to fagopyritol A1 andfagopyritol B1 with one less hydroxyl for TMS-derivatization resultingin shorter retention times. Synthesis of the insulin mediator can thenbe optimized in order to obtain appreciable amounts of the compound.Depending on efficiency, carbon-Celite columns, TLC, HPLC, or Dowex ionexchange columns can be used to purify the insulin mediator (and itsisomeric form) from the reaction mixture. The purified insulin mediatorcan then be lyophilized to a white powder. The structure of the purifiedinsulin mediator can be determined by ¹H-NMR and ¹³C-NMR (Obendorf etal., Carbohydrate Research 328:623-627 (2000); Steadman et al.,Carbohydrate Research 331:19-25 (2001), which are incorporated herein byreference in their entirety), to confirm the successful biosynthesis ofthe insulin mediator. Similarly, substituting L-chino-inositol,scyllo-inositol, or bornesitol (or other cyclitols reactive with theFeGolS-2 enzyme) in the reaction (FIG. 28) would form products that maybe used as inhibitors of the galactosamine D-chiro-inositol insulinmediator.

Discussion

A protocol has been developed that resulted in purification of thetarget enzymes from the bacterial preparation without loss of activity.Dialysis was used to remove the enzymes from the extraction buffer andinto a solution of Mn²⁺ ions. This change retained enzyme activitythroughout the purification procedure. Also, adjusting bacterial growthtimes and preparation methods further maximized the expression system.

Manganese concentration assays were used to determine that optimalenzyme action occurred in 5 mM Mn²⁺ solution. Results from the substratespecificity assays helped to identify the inositols the enzymes coulduse as galactosyl acceptors. myo-inositol, D-chiro-inositol,L-chiro-inositol, bornesitol and scyllo-inositol all can be used asgalactosyl acceptors by all three enzymes. The V_(max) and K_(m) hasbeen difficult to determine due to the sensitivity of the reaction.However, initial estimates of the K_(m) for the enzyme FeGolS-2 usingmyo-inositol as a substrate was 7.53 mM and the V_(max)0.0817 μM/min.Determination of the V_(max) and K_(m) has proven difficult for thesynthesis of fagopyritols because there are multiple products producedin the reaction.

Reactions to synthesize UDP-galactosamine and purification of thecompound have been completed (FIG. 27). UDP-galactosamine can then beused as the galactosyl donor in the reaction synthesizing the putativeinsulin mediator (FIG. 28).

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed:
 1. A method for producing a fagopyritol, an insulinmediator, an insulin mediator analogue, an insulin mediator homologue,or an insulin mediator inhibitor comprising: expressing a nucleic acidencoding a fagopyritol synthase comprising the amino acid sequence ofSEQ ID NO:4 using an expression system, said expression system having atleast a portion heterologous to the nucleic acid; providing a galactosyldonor and a galactosyl acceptor; and combining the fagopyritol synthasewith the galactosyl donor and the galactosyl acceptor under conditionseffective to produce a fagopyritol, an insulin mediator, an insulinmediator analogue, or an insulin mediator homologue.
 2. The methodaccording to claim 1, wherein the galactosyl donor is UDP-galactose. 3.The method according to claim 1, wherein the galactosyl donor isUDP-galactosamine.
 4. The method according to claim 1, wherein thegalactosyl acceptor is selected from the group consisting ofD-chiro-inositol, L-chiro-inositol, myo-inositol, bornesitol, andscyllo-inositol.
 5. The method according to claim 4, wherein thegalactosyl acceptor is D-chiro-inositol.
 6. The method according toclaim 1, wherein the fagopyritol synthase is purified.